CN108210503A

CN108210503A - Mannose is for improving the new application of Treg cell quantities and its Foxp3 factor expressions level

Info

Publication number: CN108210503A
Application number: CN201611133780.2A
Authority: CN
Inventors: 高尚先
Original assignee: Individual
Current assignee: Individual
Priority date: 2016-12-10
Filing date: 2016-12-10
Publication date: 2018-06-29
Also published as: WO2018103723A1

Abstract

This application involves drug, food, health products and cosmetic fields, specifically, are related to the new application of mannose.This application involves application of the mannose in drug, food, health products or the cosmetics for being used to prepare treatment autoimmune disease；Further relate to application of the mannose in drug, food, health products or the cosmetics for safeguarding immune homeostasis are used to prepare；Further relate to application of the mannose in the drug for improving Treg cellular levels, food, health products or cosmetics are used to prepare.Application it has been investigated that, mannose is when Treg cellular levels occur unbalance, it has certain effect to the adjustment tool of Treg cellular levels, so as to use it for the unbalance drug of Treg cellular levels, food, health products or cosmetics, to the autoimmune disease by the unbalance initiation of Treg cellular levels and by the unbalance effect with treatment and auxiliary treatment of the immune homeostasis of the unbalance initiation of Treg cellular levels.

Description

Mannose is horizontal for improving Treg cell quantities and its Foxp3 factor expressions New application

Technical field

This application involves drug, food, health products and cosmetic field, specifically, it is related to mannose for improving The new application of Treg cell quantities and Foxp3 its factor expression levels.

Background technology

Immune function (immunologic function) is that body is thin in lymphocyte, monocyte, macrophage, NK It is completed under born of the same parents and other interactions in relation to cell and its product；Immune function is that immune system is sent out according to Immune discrimination The effect waved.The immune function of body is mainly manifested in three aspects i.e. immunoprophylaxis, immune stabilization and immunosurveillance.

1st, immunoprophylaxis (immunoligic defence) refers to body and resists and remove pathogenic microorganism or other foreign matters Function.Immunoprophylaxis function, which is abnormal, can cause disease, such as react excessively high and may occur in which hypersensitivity；Reacting too low causes to exempt from Epidemic disease defect disease.

2nd, it is immunized and stablizes (immunoligic hemeostasis)：Refer to the cell that body removes damage or aging, maintain it The function of physiological equilibrium.Immunologic homeostasis imbalance can lead to autoimmunity disease.

3rd, immunosurveillance (immunoligic surveillance)：It is thin to refer to the mutation that body identification and removing occur in vivo Born of the same parents prevent the function of tumour.Immune surveillance function is low, is susceptible to suffer from malignant tumour.

Wherein, stable (immune homeostasis) is immunized to refer between the cell composition of immune system, immunocyte, carefully Interaction between born of the same parents and cytokine network maintains stablizing relatively for its functional status.Autoimmunity disease (autoimmune Diseases) refer to that body occurs immune response to autoantigen and leads to the disease caused by damaged self tissue.

Immune system disorder plays an important role in morbidity, and immunological regulation is increasingly becoming the center ring of autoimmunity disease Section.More and more the study found that regulatory T cells (regulatory T cell, Treg) are in physiology and pathophysiological condition Under play a key effect to immunological regulation, played an important role in infection, tumour, trnasplantion immunity and autoimmunity disease. Treg is broadly divided into two major class：Natural regulation T cell (natural Treg, nTreg) and inductivity regulatory T cells (inducedTreg, iTreg).Treg is a kind of CD4 with immunosuppressive activity⁺T cell subgroup, Treg cells are by releasing It puts cell factor IL-10 and TGF-β plays the function of inhibiting T cell and antigen presenting cell, reduce the production of inflammatory cytokine Raw and antibody-secreting and play immunological effect.Foxp3 is the important transcription factor of Treg, and continuous expression is that Treg is maintained to inhibit The key factor of activity.Treg is by raising the expression of inhibition immunocyte surface molecular and lowering the dependency basis of activating T cell Because of expression, immunoregulation effect is played.Initial CD4⁺T cell by exotic antigen after being stimulated, in the effect of costimulatory signal Lower activation is divided into the effector T cell of different subtype.Initial CD4⁺T cell can be divided under the independent induction of TGF-β Treg cells when IL-6 is existed simultaneously, can induce the expression of ROR γ t, Treg cells is inhibited to generate, promote initial CD4⁺T Cell differentiation is into Thl7 cells.

When immune homeostasis gets muddled, Treg cells and other immunocytes such as Thl7 cell quantities level are unbalance, lead to Cross raising Treg cell quantities and its Foxp3 factor expressions horizontal adjustment Treg cells and other immunocytes such as Thl7 cell numbers Amount level provides new way for treatment immune homeostasis disorder.In consideration of it, special propose the application.

Invention content

The primary goal of the invention of the application be propose mannose be used to prepare treatment autoimmune disease drug, Application in food, health products or cosmetics.

The second goal of the invention of the application is to propose that mannose is being used to prepare drug, food for safeguarding immune homeostasis Application in product, health products or cosmetics.

The third goal of the invention of the application is to propose that mannose is being used to prepare raising Treg cell quantities and its Foxp3 Application in the drug of factor expression level, food, health products or cosmetics.

In order to complete the purpose of the application, the technical solution used for：

This application involves mannose in drug, food, health products or the makeup for being used to prepare treatment autoimmune disease Application in product.

Preferably, the autoimmune disease is autoimmune disease caused by being reduced due to Treg cell quantities.

Preferably, the autoimmune disease is selected from lupus erythematosus, hyperthyroidism, I types or type-2 diabetes mellitus And its complication, IgA ephritis, mouth xerophthalmia scheorma syndrome, rheumatoid arthritis, ankylosing spondylitis, simple obesity, branch gas Pipe asthma, neurodermatitis, canker sore, ulcerative colitis, ox-hide moss, leucoderma, Behcet's disease, autoimmune are wet Rash, Autoimmune uveitis, autoimmune eye iris cyclitis, autoimmunity membranous conjunctivitis, autoimmune are done Eye disease, autoimmune cataract, autoimmune glaucoma, allergic rhinitis, irritable bowel syndrome, pruitus.

The application further relates to mannose and is being used to prepare drug, food, health products or makeup for safeguarding immune homeostasis Application in product.

Preferably, the disease as caused by the immune system homeostasis is selected from lupus erythematosus, hyperthyroidism, I Type or type-2 diabetes mellitus and its complication, IgA ephritis, mouth xerophthalmia scheorma syndrome, rheumatoid arthritis, ankylosing spondylitis, Simple obesity, bronchial asthma, neurodermatitis, canker sore, ulcerative colitis, ox-hide moss, leucoderma, Behcet Disease, autoimmune eczema, Autoimmune uveitis, autoimmune eye iris cyclitis, autoimmune conjunctiva Inflammation, autoimmune xerophthalmia, autoimmune cataract, autoimmune glaucoma, allergic rhinitis, intestines easily swash synthesis Sign, pruitus.

Wherein, in this application autoimmune xerophthalmia refer to by autoimmunity sexual factor influence caused by or by Xerophthalmia caused by autoimmune disease；Autoimmune cataract refer to by autoimmunity sexual factor influence caused by, Or as the cataract caused by autoimmune disease；Autoimmune glaucoma refers to influence institute by autoimmunity sexual factor Cause or as the glaucoma caused by autoimmune disease.

The application further relates to mannose in the medicine for being used to prepare raising Treg cell quantities and its Foxp3 factor expressions level Application in object, food, health products or cosmetics.

Mannose in above application is preferably D-MANNOSE.

The technical solution of the application at least has following beneficial effect：

The application it has been investigated that, mannose occurs in Treg cells and other immunocytes such as Thl7 cell quantities level When unbalance, to increasing Treg cell quantities and its Foxp3 factor expressions level and then adjusting Treg cells and other immunocytes It is thin so as to use it for Treg cells and other immunocytes such as Thl7 if Thl7 cell quantities level tool has certain effect Drug, food, health products or the cosmetics that born of the same parents' quantity levels are unbalance, to by Treg cells and other immunocytes such as Thl7 cells The autoimmune disease for initiation that quantity levels are unbalance and by Treg cells and other immunocytes such as Thl7 cell quantities water Equal the unbalance effect with treatment and auxiliary treatment of immune homeostasis of unbalance initiation.

Description of the drawings：

Fig. 1 is the preparation flow of antigen protein used in dry syndrome animal model.

With reference to specific embodiment, the application is expanded on further.It should be understood that these embodiments are merely to illustrate the application Rather than limitation scope of the present application.

Specific embodiment

This application involves mannose in drug, food, health products or the makeup for being used to prepare treatment autoimmune disease Application in product.Mannose in the application is preferably D-MANNOSE.

In this application, autoimmune disease (autoimmune diseases) refers to that autoantigen occurs for body Immune response and lead to the disease caused by damaged self tissue, include lupus erythematosus, hyperthyroidism, due to Treg I types or type-2 diabetes mellitus caused by cell quantity reduces and its complication, IgA ephritis, mouth xerophthalmia scheorma syndrome, rheumatoid close Save inflammation, ankylosing spondylitis, due to Treg cell quantities reduction caused by simple obesity, bronchial asthma, neurodermatitis, Canker sore, ulcerative colitis, ox-hide moss, leucoderma, Behcet's disease, autoimmune eczema, Autoimmune uveitis film Inflammation, autoimmune eye iris cyclitis, autoimmunity membranous conjunctivitis, allergic rhinitis.And when generation autoimmune disease When sick, and the application can promote Treg thin in Treg cells under pathological state when mannose by finding with stimulation Born of the same parents' level improves, and so as to adjust the balance of Treg cells and Th17 cells, immune stabilization is safeguarded, so as to autoimmune Disease plays treatment and auxiliary therapeutic action.

In the drug for being used to prepare treatment autoimmune disease, mannose can be prepared into a variety of dosage forms, such as oral Preparation, ejection preparation and external preparation, oral preparation are selected from the dosage forms such as tablet, buccal tablet, disintegrant, oral liquid, capsule, injection Preparation may be selected from liquid drugs injection or freeze drying powder injection, external preparation may be selected from external application aqua, washing lotion, gel, spray, patch, eye drops, Paste or spit of fland agent.

In the food for being used to prepare treatment autoimmune disease, mannose can be prepared into numerous food, such as soft sweets, firmly Sugar, cake, jelly, beverage etc..

In the health products for being used to prepare treatment autoimmune disease, mannose can be prepared into a variety of dosage form health products, Such as tablet, buccal tablet, disintegrant, oral liquid, capsule, oral liquid.

In the cosmetics for being used to prepare treatment autoimmune disease, mannose can be prepared into the makeup of variform Product, such as aqua, washing lotion, gel, spray, patch, paste.

In the drug, food, health products or the cosmetics that prepare treatment autoimmune disease, the dosage of mannose is 0.001~200g/ days.

The application further relates to mannose and is being used to prepare drug, food, health products or makeup for safeguarding immune homeostasis Application in product.Through recent studies have found that, lupus erythematosus, hyperthyroidism, I types or type-2 diabetes mellitus and its complication, Chronic ulcerative colitis, IgA ephritis, mouth xerophthalmia scheorma syndrome, rheumatoid arthritis, ankylosing spondylitis, pure fertilizer Fat, bronchial asthma, canker sore, ulcerative colitis, ox-hide moss, leucoderma, Behcet's disease, itself is exempted from neurodermatitis Epidemic disease eczema, autoimmune eye iris cyclitis, autoimmunity membranous conjunctivitis, itself is exempted from Autoimmune uveitis Epidemic disease uveitis, autoimmune eye iris cyclitis, autoimmunity membranous conjunctivitis, autoimmune xerophthalmia, itself Immune cataract, autoimmune glaucoma, allergic rhinitis, irritable bowel syndrome, pruitus.Wherein, autoimmunity Property xerophthalmia refer to by autoimmunity sexual factor influence caused by or as the xerophthalmia caused by autoimmune disease；From Body be immunized cataract refer to it is caused by autoimmunity sexual factor influences or as in white caused by autoimmune disease Barrier；Autoimmune glaucoma refer to by autoimmunity sexual factor influence caused by or as caused by autoimmune disease Glaucoma.These diseases are as caused by getting muddled immune homeostasis.Mannose can by adjusting the levels of Treg cells, Immune homeostasis is safeguarded, so as to play treatment and auxiliary therapeutic action to these diseases.

When being used to prepare the drug for safeguarding immune homeostasis, mannose can be prepared into a variety of dosage forms, such as oral system Agent, ejection preparation and external preparation, oral preparation are selected from the dosage forms such as tablet, buccal tablet, disintegrant, oral liquid, capsule, injection system Agent may be selected from liquid drugs injection or freeze drying powder injection, and external preparation may be selected from external application aqua, washing lotion, gel, spray, patch, eye drops, cream Agent or spit of fland agent.When being used to prepare the food for safeguarding immune homeostasis, mannose can be prepared into numerous food, such as soft sweets, firmly Sugar, cake, jelly, beverage etc..

When being used to prepare the health products for safeguarding immune homeostasis, mannose can be prepared into a variety of dosage form health products, as tablet, Buccal tablet, disintegrant, oral liquid, capsule, oral liquid etc..

When being used to prepare the cosmetics for safeguarding immune homeostasis, mannose can be prepared into the cosmetics of variform, such as water Agent, washing lotion, gel, spray, patch, paste etc..

It prepares in drug, food, health products or cosmetics of the treatment for safeguarding immune homeostasis, the dosage of mannose is 0.001~200g/ days.

The Foxp3 factors are the members of plug shape transcription factor family, for Treg cell developments and the crucial tune functioned Save the factor.After can be by being combined with chromosome, expression and the function of several genes be adjusted, and then control the development of Treg cells And function.Research shows that Foxp3 is CD4⁺CD 25⁺Treg develops and the key molecule of function, the CD4⁺Treg can inhibit TCR Traditional C D4 of mediation⁺T cell is proliferated and generates cell factor, is maintaining peripheral tolerance, is preventing from sending out in autoimmune disease Wave important function.Obtain (gain of function) experimental study by some functions, directly demonstrate Foxp3 with it is natural The relationship of Treg cells, it is required that the expression of Foxp3 obtains Treg cell phenotypes and function for nave T cell, passes through work( (loss of function) experiment can be lost and show that Foxp3 is that Treg cell developments are indispensable.Foxp3 is in Treg cells Development and be functionally required, and Foxp3 is only in CD4⁺CD25⁺It is expressed in T cell, in natural CD4⁺CD 25^-T cell and It is not expressed in the thymocyte of non-maturation, in B cell and CD8⁺Also it can regard Foxp3 as Treg without apparent expression in T cell The Specific marker of cell regards Foxp3 positive T cells as Treg cells.Mannose can be by improving Treg cell quantities And its Foxp3 factor expressions are horizontal, safeguard immune homeostasis, so as to horizontal by Treg cell quantities and its Foxp3 factor expressions Disease caused by decline plays treatment and auxiliary therapeutic action.

When being used to prepare the drug for improving Treg cell quantities and its Foxp3 factor expressions level, mannose can be prepared Into a variety of dosage forms, such as oral preparation, ejection preparation and external preparation, oral preparation is selected from the agent such as tablet, oral liquid, capsule Type, ejection preparation may be selected from liquid drugs injection or freeze drying powder injection, and external preparation may be selected from external application aqua, paste or spit of fland agent.

In the food for being used to prepare the expression for improving Treg cell quantities and the Foxp3 factors, mannose can be prepared Into numerous food, such as soft sweets, hard candy, cake, jelly, beverage.

In the health products for being used to prepare the expression for improving Treg cell quantities and the Foxp3 factors, mannose can be made It is standby into plurality kinds of health care product, such as tablet, buccal tablet, disintegrant, oral liquid, capsule, oral liquid.

In the cosmetics for being used to prepare the expression for improving Treg cell quantities and the Foxp3 factors, mannose can be made The standby cosmetics into variform, such as aqua, washing lotion, gel, spray, patch, paste.

It prepares in drug, food, health products or cosmetics that treatment improves Treg cellular levels, the dosage of mannose is 0.001~200g/ days.

It has been found that D-MANNOSE in a variety of animal models for causing Th17/Treg cellular levels unbalance, has and carries High Treg cellular levels maintain the effect of immune homeostasis.Such as in rheumatoid arthritis, lupus erythematosus, bronchial asthma, sugar Urinate disease, obesity, hyperthyroidism, IgA ephritis, mouth xerophthalmia scheorma syndrome, neurodermatitis, canker sore, exedens knot Enteritis, ox-hide moss, autoimmune eczema, Autoimmune uveitis, immune membranous conjunctivitis, xerophthalmia, allergic rhinitis, In the animal models such as irritable bowel syndrome, cataract, after the animal pattern D-MANNOSE of 7~28 days is given, Treg cellular waters It is flat to be improved, and the clinical symptoms of animal pattern have obtained a degree of alleviation.Below into one by taking animal model as an example Step illustrates the technique effect of the application.

Embodiment 1：Bronchial asthma animal model experiment

1 materials and methods

The foundation of 1.1 animal packets and asthmatic model

Cleaning grade Balb/c mouse are purchased from Shandong University's Experimental Animal Center, raise and are raised in box in Special sterilizing, constant Room temperature (21~25 DEG C) crosses air filtering, and humidity 50%~65%, 10~12h/d of illumination, feed, water are all sterile-processed, replaces The operations such as feed carry out in super-clean bench, and whole experiment process is carried out by animal protection and instruction policy.

Mouse is randomly divided into control group, asthma group and treatment group (every group 10)：

(1) asthma group gives egg protein (OVA) alum adjuvant (Sigma companies) 200 μ l abdominal cavities for the 6th week, the 8th week in mouse Sensitization is injected, 1% egg protein (OVA) High frequency aerosol inhalation is given (i.e. 1 week after the 2nd sensitization) within the 9th week and is excited, daily 1 It is secondary, each 30min, continuous 7d.Carry out model verification and Cell counts.

(2) same asthma group is tested in the modeling for the treatment of group mouse, after asthma group verification modeling success, feeds D-MANNOSE water (1.0M) is freely drunk, and after continuous 21 days, carries out Cell counts.

(3) same time of the control group in modeling experimentation gives simple alum adjuvant (without OVA) sensitization and PBS Neulized inhalation excites.

1.2 bronchoalveolar lavage

Mouse anesthesia is put to death, dorsal position fixes exposure tracheae, trachea cannula and fixation is carried out with remaining needle, with cold PBS Lavation, each 0.5ml, common 1ml are centrifuged after irrigating solution cell count, and cell sediment is taken to carry out Wright's staining classified counting of leucocyte.

1.3 lung tissue Foxp3⁺Treg and Th17 cell detections

With PBS from mouse right ventricle be perfused, remove Pulmonary Vascular blood, Isolated-lung tissue shred after 1ml contain 0.002g (0.2%) 15min is incubated in the PBS of Collagenase I (Sigma-Aldrich), lung tissue is placed in 300 mesh stainless steel mesh It above and with eye scissors is shredded, collects single cell suspension in culture dish, 2500r/min centrifugation 5min remove supernatant, add in 2ml Erythrocyte cracked liquid is incubated at room temperature 8min, and PBS washes 2 centrifugations and removes supernatant, then will with 1640 culture mediums containing 10% fetal calf serum Single cell suspension is made in cell, adds in 50ng/ml PMA (Sigma-Aldrich), 500ng/ml ionomycins (Sigma- Aldrich) and 0.7 μ l/ml GolgiPlug (BD Biosciences) are in 37 DEG C, 5%CO₂4~6h is incubated in incubator jointly.

It collects the centrifugation of the cell after being incubated and removes supernatant, fluorescent labeled antibody is added in after mixing, adds in Hamster Antimouse CD3-Pecy7 (BD Biosciences), Ratantimouse CD4-FITC (BD Biosciences), room Temperature, which is protected from light, is incubated 30min, washes 2 centrifugations with PBS and removes supernatant, fixative fixes 30min, and PBS washes 1 centrifugation and removes supernatant, adds in Supernatant is removed in the incubation at room temperature 5min centrifugations of penetrating liquid, after mixing plus Rat antimouse IL-4-APC (BDBiosciences), Rat antimouse IL-17-PE(BDBiosciences)、Rat antimouse IFN-γ-PerCPcy5.5 (BDBioscience), room temperature, which is protected from light, is incubated 30min, and centrifugation goes after supernatant after adding in 200 μ l PBS mixings to use flow cytometer (FACSClibur, BDBiosciences) is detected.

Foxp3⁺The detection of Treg cells：Add in Rat antimouse CD4-FITC (BDBiosciences) Rat Antimouse CD25-PE (BDBiosciences) room temperature, which is protected from light, is incubated 30min, washes 2 times with PBS and removes supernatant, fixative is fixed 30min is washed 1 time with PBS, adds in penetrating liquid 5min, adds antimouse Foxp3-PEcy5 (eBBiosciences), room temperature is kept away Light is incubated 30min, cell flow cytometer type (FAC-SClibur, the BDBiosciences) detection after label.

1.3 statistical analysis data are represented with mean ± standard deviation, using Graphad prism5.0 statistical softwares, two groups Between mean compare and examined using t, with P<0.05 is statistically significant for difference.

2 results

2.1 mouse bronchial bronchoalveolar lavage fluid cell counts and differential counting comparison result

The results are shown in Table 1, in normal mouse BAL fluid be mainly macrophage and lymphocyte, especially with Based on the former, and eosinophil and neutrophil content are very few.Cell in asthma group mouse bronchial bronchoalveolar lavage fluid Sum, lymphocyte, eosinophil, neutrophil leucocyte ratio are obviously higher than control group (P<0.001) OVA groups, are prompted Airway of mice inflammation is apparent compared with control group.Show modeling success.

Table 1：Bronchoalveolar Lavage Fluid Cells sum and leukocyte differential count

* P compared with the control group<0.001.

2.2 mouse lung tissue Foxp3⁺Treg, Th17 cell, Foxp3⁺Treg/Th17 testing results

The results are shown in Table 2：

Table 2：

Cell	Control group	Asthma group	Treatment group
				Treg cell percentages	(7.67 ± 0.44) %	(4.87 ± 0.35) %	(6.87 ± 0.65) %^Δ
Th17 cell percentages	(1.07 ± 0.07) %	(1.74 ± 0.17) %*	(1.21 ± 0.08) %^Δ
				Treg/Th17	7.38±0.71	3.02±0.49**	5.68±0.67^Δ

* P compared with the control group<0.01；* P compared with the control group<0.01；Δ P compared with asthma group<0.01.

Find that after to animal model application D-MANNOSE, Treg cellular levels obtain according to bronchial asthma animal model To raising, at the same the clinical symptoms for the treatment of group also improve to a certain extent, while its leucocyte level is also remarkably decreased.Experiment Example 2：Arthritis animal model is tested

1 materials and methods

The foundation of 1.1 animal packets and CIA animal models (collagen-induced rat arthritis model)

Wistar rats (male), weight 100g or so, age of mouse 4 weeks, purchased from Shandong University's Experimental Animal Center.Raise item Part is carried out with embodiment 1, whole experiment process by animal protection and instruction policy.Rat is randomly divided into control group, CIA Model group and treatment group (every group 20)：

(1) CIA model groups：By 7ml ox II Collagen Type VIs, (collagen type II, CII contains acetic acid, a concentration of before experiment 2rng/ml), it is slowly added dropwise to isometric Freund's complete adjuvant (complete freund's adjuvant, CFA, Sigma Company) in fully emulsified, the final concentration of 1mg/ml of II Collagen Type VIs.By the collagen prepared and adjuvant mixture in rat root of the tail Portion, intracutaneous injection, the 0th day 200 μ g (0.2ml) of initial immunity, the 7th day in 200 μ g (0.2ml) of offside root of the tail portion booster immunization.

Initial immunity：For RA model groups to the emulsion 0.2ml containing ox II Collagen Type VIs 1mg/ml is given, Normal group is big The physiological saline of mouse intracutaneous injection equivalent.

Booster immunization：After initial immunity animal 1 week, RA model groups give the emulsion containing ox II Collagen Type VIs Img/ml 0.2ml, the physiological saline of rats in normal control group intracutaneous injection equivalent.

Arthritis index (arthritis index, AI) evaluation is carried out after initial immunity weekly：0 point=without swelling or have The arthritis such as erythema show；Joint involvement between 1 point=1-2 happiness；Joint or 1 bigger joint involvement between 2 points=3-4 happiness；3 Point=more than 4 joint involvements；The serious arthritis of 4 points=full pawl；Divide per pawl 4,16 points altogether.Because after lesion is mainly involved Foot so 6-8 points are severe arthritic, is observed 1 time before initial immunity, observation one in every 3 days 6 weeks before acute stage after initial immunity It is secondary, hereafter chronic phase observe weekly 1 time.9~21 days after initial immunity rat, there are 42 in 45 wistar rats of experimental group Modeling success, for subsequent experimental.

(2) treatment group：By the successful rat of modeling 20, feed D-MANNOSE water (1.0M) and freely drink, after continuous 21 days, Carry out Cell counts.

(3) same time of the control group in modeling experimentation gives physiological saline.

1.2 spleen tissue Foxp3⁺Treg and Th17 cell detections

Rats by intraperitoneal injection overdose of sodium pentobarbital is put to death, spleen is taken under aseptic condition, is used after being rinsed with PBS buffer solution Eye scissors are cut into small pieces, and in grinding filtration on 200 mesh steel sieve, 2000r/min centrifugation 5min abandon supernatant, add in erythrocyte splitting Liquid 5mL mixings, stand 5min, and 2000r/min centrifugation 5min abandon supernatant, PBS buffer solution is washed 1~2 time, obtains splenocyte.

It is detected by method in embodiment 1 using flow cytometer.

2 result testing results are as shown in table 3：

Table 3：

Note：* P compared with the control group<0.01；* P compared with the control group<0.01；Δ P compared with model group<0.01.

Find that after to animal model application D-MANNOSE, Treg cellular levels are improved according to CIA animal models, The clinical symptoms for the treatment of group also improve to a certain extent.

By the animal experimental model simultaneously also it can be confirmed that D-MANNOSE is to ankylosing spondylitis (poker back) Also there is certain therapeutic effect.

Experimental example 3：NOD animal model experiments

1 materials and methods

1.1 animal packet

Non-obese patients with type Ⅰ DM (nonobese diabetic, the NOD) mouse (40) of 8 week old female, weight (20.0 ± 2.0) g is bought from Shanghai Si Laike Experimental Animal Centers.It raises and is raised in box in Special sterilizing, constant room temperature (21~25 DEG C), Air filtering is crossed, humidity 50%~65%, 10~12h/d of illumination, feed, water are all sterile-processed, replace the operations such as feed and exist It is carried out in super-clean bench, whole experiment process is carried out by animal protection and instruction policy.

NOD mouse are randomly divided into control group, treatment group (every group 20)：D-MANNOSE water (1.2M) is fed by treatment group, even After 21 days continuous, Cell counts are carried out.

1.2 spleen tissue Foxp3⁺Treg and Th17 cell detections

Mouse boosting tissue's cell is taken using method in embodiment 2, is detected using flow cytometer.

2 result testing results are as shown in table 4：

Table 4：

Cell	NOD model groups	Treatment group
			Treg cell percentages	(0.34 ± 0.03) %	(0.48 ± 0.12) %^Δ
Th17 cell percentages	(0.90 ± 0.15) %	(0.70 ± 0.21) %^Δ
			Treg/Th17	0.38±0.12	0.68±0.18^Δ

Note：Δ P compared with model group<0.01.

Find that after to animal model application D-MANNOSE, Treg cellular levels are improved according to NOD animal models, The clinical symptoms for the treatment of group also improve to a certain extent.

Embodiment 4：Animal Model of Ulcerative Colitis is tested

1 materials and methods

The foundation of 1.1 animal packets and UC animal models (Ulcerative Colitis Model)

Healthy adult Sprague-Dawley (SD) rat, half male and half female, 200g or so, purchased from Shandong University experimental animal Center, rearing conditions are carried out with embodiment 1, whole experiment process by animal protection and instruction policy.

Rat is randomly divided into control group, UC model groups and treatment group (every group 20)：

(1) model group：

50g DSS (dextran sulfate sodium) are added in 1000ml distilled water, are fully dissolved, are made into 5%DSS solution, often Day according to the actually amount of drinking Fresh, freely drinks 1 week for rat.Rat hair color dries up, and spirit owes active, reacts and owes spirit It is living, started the symptoms such as diarrhea, bloody stool, weight mitigation occur at the 2nd~4 day.

1.2 spleen tissue Foxp3⁺Treg and Th17 cell detections

Mouse boosting tissue's cell is taken to be detected using method in embodiment 3, be detected using flow cytometer.

2 result testing results are as shown in table 5：

Table 5：

Cell	Control group	UC model groups	Treatment group
				Treg cell percentages	(0.43 ± 0.05) %	(0.31 ± 0.23) %**	(0.39 ± 0.12) %^Δ
Th17 cell percentages	(0.51 ± 0.03) %	(0.89 ± 0.12) %**	(0.65 ± 0.21) %^Δ
				Treg/Th17	0.84±0.01	0.35±0.14**	0.60±0.18^Δ

Find that after to animal model application mannose, Treg cellular levels are improved according to UC animal models, treatment Group clinical symptoms also improve to a certain extent.

Embodiment 5：Lupus erythematosus animal model experiment

1 materials and methods

1.1 animal packet

The spontaneous systemic loupus erythematosus lupoid acne model mices of SPF grades of MRL/lpr 40, female, weight 20g or so, March Age, purchased from Shandong University's Experimental Animal Center.Experiment whole process is in the enclosed grazing of SPF Ji Yan animal housing.Mouse is randomly divided into pair According to group and treatment group (every group 20)：

(1) treatment group：It feeds mouse to D-MANNOSE water (1.0M) freely to drink, after continuous 21 days, carries out Cell counts.

(2) same time of the control group in modeling experimentation gives physiological saline.

1.2 spleen tissue Foxp3⁺Treg and Th17 cell detections

2 result testing results are as shown in table 6：

Table 6：

Cell	Model group	Treatment group
			Treg cell percentages	(0.32 ± 0.04) %	(0.41 ± 0.10) %^Δ
Th17 cell percentages	(0.91 ± 0.07) %	(0.70 ± 0.22) %^Δ
			Treg/Th17	0.35±0.13	0.58±0.16^Δ

Note：Δ P compared with model group<0.01.

It is found according to spontaneous systemic loupus erythematosus lupoid acne model, after to animal model application D-MANNOSE, Treg Cellular level is improved, at the same the clinical symptoms for the treatment of group also improve to a certain extent.

Embodiment 6：Animal Models of Psoriasis is tested

1 materials and methods

The foundation of 1.1 animal packets and Animal Models of Psoriasis

Cleaning grade Balb/c mouse are purchased from Shandong University's Experimental Animal Center, raise in Special sterilizing raising box, raise Condition is carried out with embodiment 1, whole experiment process by animal protection and instruction policy.

Mouse is randomly divided into control group, model group and treatment group (every group 20)：

(1) model group：

After mouse electric shaver cropping, remaining the every mouse in addition to blank group is applied to 5% imiquimod cream Back baring skin, 50mg/ only, establish Animal Models of Psoriasis on 1/th.Compared with Normal group, imiquimod cream is smeared There is erythema in model group skin after 1d；There is the scales of skin that peel off, 4~5d most serious in 2~3d；6d skin lesions are slightly better, and 7~8d skins are again It aggravates and thickens.Pathological section shows that hyperkeratinization, parakeratosis, polymorphonuclear cell leaching in acanthosis and cuticula occurs in skin The Histological change of similar mankind's psoriasis such as profit.

(2) treatment group：It feeds modeling success mouse to D-MANNOSE water (1.0M) freely to drink, after continuous 21 days, carry out thin Born of the same parents count.

1.2 spleen tissue Foxp3⁺Treg and Th17 cell detections

2 result testing results are as shown in table 7：

Table 7：

Cell	Control group	Model group	Treatment group
				Treg cell percentages	(0.40 ± 0.05) %	(0.30 ± 0.23) %**	(0.38 ± 0.12) %^Δ
Th17 cell percentages	(0.50 ± 0.03) %	(0.90 ± 0.12) %**	(0.67 ± 0.21) %^Δ
				Treg/Th17	0.80±0.04	0.30±0.19**	0.57±0.18^Δ

It is found according to psoriasis model, after to animal model application D-MANNOSE, Treg cellular levels are improved, and are controlled The clinical symptoms for the treatment of group also improve to a certain extent.

Embodiment 7：Recurrent aphthous ulcer animal model experiment

1 materials and methods

1.1 animal packets and RAU (recurrent aphthous ulcertion, recurrent aphthous ulcer) animal model are built It is vertical

Wistar rats, half male and half female, 180~200g of weight, rearing conditions with embodiment 1, press by whole experiment process Object is protected and instruction policy carries out.

Rat is randomly divided into control group, model group and treatment group (every group 20)：

(1) model group：

Mucous membrane of mouth will be stripped after 30 Wistar rat anesthesias, adds in 0.1mol/L, the PBS buffer solution of pH7.4 is made Tissue -60 DEG C of low temperature refrigerators of homogenate save backup.During immune animal, above-mentioned tissue is homogenized and is mixed with Freund's adjuvant equal proportion, It is operated by adjuvant specification and antigen emulsifier (not stratified after placement) is made.

40 rat (half male and half female) backbone both sides are cut off into mouse hair, 0.1mL antigens are subcutaneously injected per side after iodophor disinfection Emulsifier is injected 1 time weekly, co-injection 6 times.Observe the variation of rat oral mucosa.Experimental rat is the 2nd after last time is injected Its mucous membrane of mouth starts contrafluxion, and ulcer occur in the 6th day all experimental rats after injection, and ulcer mostly occurs in lip, cheek Mucous membrane, mouth bottom, ulcer is oval or round, neat in edge, and there is lark pseudomembrane on surface, and diameter 1~2mm, general 3~5d are certainly Row healing, it is rear to recur again.Show modeling success,

(2) treatment group：It feeds modeling success rat to D-MANNOSE water (1.2M) freely to drink, after continuous 21 days, carry out thin Born of the same parents count.

(3) another 20 non-injections of antigens rats are Normal group.

1.2 spleen tissue Foxp3⁺Treg and Th17 cell detections

Rat spleen histocyte is taken to be detected using method in embodiment 3, be detected using flow cytometer.

2 result testing results are as shown in table 8：

Table 8：

Cell	Control group	RAU model groups	Treatment group
				Treg cell percentages	(0.46 ± 0.04) %	(0.30 ± 0.23) %**	(0.41 ± 0.14) %^Δ
Th17 cell percentages	(0.53 ± 0.05) %	(0.97 ± 0.11) %**	(0.70 ± 0.13) %^Δ
				Treg/Th17	0.87±0.01	0.31±0.07*	0.59±0.06^Δ

Find that after to animal model application D-MANNOSE, Treg cellular levels are improved according to RAU models, treatment Group clinical symptoms also improve to a certain extent.

Embodiment 8：Diet-induced obesity animal model experiment

1 materials and methods

The foundation of 1.1 animal packets and Diet-induced obesity (DIO) animal model

50 healthy SPF grades of male SD rats just weaned, 40~50g of weight, purchased from Shandong University's Experimental Animal Center. Rearing conditions are carried out with embodiment 1, whole experiment process by animal protection and instruction policy.

Rat is randomly divided into control group, model group：

(1) model group：50, high-energy high fat diet is given, feeds 14 weeks, measures rat body weight on every Mondays, is observed Each group rat body weight changes.After 14 weeks, highest 20 rats of high energy feed group the weight of animals are set as DIO groups；Take 10 DIO Rat carries out Cell counts.

(2) treatment group：Modeling success 10 hello D-MANNOSE water (1.0M) of rat are freely drunk, after continuous 21 days, into Row Cell counts.

(3) 10 rats of control group give basic rat feed.

Wherein, standard animal feed components：5% fat, 55% carbohydrate, 22% protein, 7% ash content, 5% is fine Dimension element.Feed heat is 3.80kcal/g.

High energy feed composition：30% fat, 40% carbohydrate, 15% protein, 4% ash content, 3% cellulose.It raises Material heat is 4.76kcal/g.

1.2 spleen tissue Foxp3⁺Treg and Th17 cell detections

1.3 statistical analysis

Data represent that, using Graphad prism5.0 statistical softwares, mean is relatively adopted between two groups with mean ± standard deviation It is examined with t, with P<0.05 is statistically significant for difference.

2 result testing results are as shown in table 9：

Table 9：

Cell	Control group	DIO model groups	Treatment group
				Treg cell percentages	(0.44 ± 0.03) %	(0.38 ± 0.23) %**	(0.40 ± 0.35) %^Δ
Th17 cell percentages	(0.49 ± 0.02) %	(0.92 ± 0.15) %**	(0.76 ± 0.32) %^Δ
				Treg/Th17	0.90±0.03	0.41±0.02*	0.52±0.23^Δ

Find that after to animal model application D-MANNOSE, Treg cellular levels are improved according to DIO models, treatment Group clinical symptoms also improve to a certain extent.

Embodiment 9：IgA nephrosis animal models are tested

1 materials and methods

The foundation of 1.1 animal packets and IgA nephrosis animal models

80 male and healthy cleaning grade Wistar rats are chosen, 200 ± 20g of weight rearing conditions are entire real with embodiment 1 Process is tested to carry out by animal protection and instruction policy.After adaptability is fed 1 week, rat is divided into control group, model group at random：

(1) model group：50, modeling group gives bovine serum albumin(BSA) (Bevine serum albumin, BSA) oral filling Stomach, 25% carbon tetrachloride (Carbon tetrachloride, CCL4) solution is subcutaneously injected and 0.25g/L lipopolysaccharides The method of (Lipopolysaccharide, LPS) tail vein injection establishes IgA nephrotic rats models, totally 9 weeks.

(3) 10 rats of control group give physiological saline.

1.2 spleen tissue Foxp3⁺Treg and Th17 cell detections

2 result testing results are as shown in table 10：

Table 10：

Cell	Control group	IgA model groups	Treatment group
				Treg cell percentages	(0.45 ± 0.03) %	(0.35 ± 0.23) %**	(0.42 ± 0.32) %^Δ
Th17 cell percentages	(0.50 ± 0.02) %	(0.96 ± 0.15) %**	(0.72 ± 0.31) %^Δ
				Treg/Th17	0.90±0.03	0.36±0.21**	0.58±0.35^Δ

It is found according to animal model experiment, after to animal model application D-MANNOSE, Treg cellular levels are improved, The clinical symptoms for the treatment of group also improve to a certain extent.

Embodiment 10：The syndrome animal model experiment of mouth xerophthalmia scheorma

1 materials and methods

1.1 Freund's complete adjuvants are prepared with antigen

Cleaning grade Balb/c mouse are purchased from Shandong University's Experimental Animal Center, raise to raise in box in Special sterilizing and raise item Part is carried out with embodiment 1, whole experiment process by animal protection and instruction policy.

By lanolin heating and melting, add in atoleine and press 1:3(v:V) after ratio is prepared, 8 pounds of 20 minutes high pressure sterilizations BCG (final concentration 6mg/ml) is added in IFA after cooling by as incomplete Freund's adjuvant (IFA), fully emulsified to get Freund Freund's complete adjuvant (CFA).

The preparation of 1.2 dry syndrome animal models

The salivary gland of Balb/c mouse is taken out, lymph node is rejected, is put into PBS buffer solution, is homogenized.After homogenate with differential from Heart method, according to flow shown in Fig. 1 from 1000g to 10⁵G will be 10⁵Isolated component is set as component P5 during g.Use spectrophotometric Meter is by Coomassie brilliant blue colorimetric method for determining P5 antigen protein contents.P5 albumen and CFA are emulsified, obtaining final antigen concentration is The emulsification P5 antigens of 0.75mg/ml.Rat is divided into control group, model group at random：

(1) model group：Emulsification P5 proteantigens, 0.1ml/ foot is subcutaneously injected in two hind paw of mouse and both sides groin It slaps, 3mg/ml containing BCG, the 1st day, the 7th day and the 21st day after first immunisation, pertussis vaccine 0.2ml (pertussis is injected intraperitoneally Dead cell concentration is 2.9 × 10¹⁰A/milliliter), after first immunisation the 21st day with the 35th day, emulsification P5 proteantigens are subcutaneously injected Booster immunization generates the salivary gland change and clinical manifestation of similar SS.

(2) treatment group：Modeling success 10 hello D-MANNOSE water (1.0M) of mouse are freely drunk, after continuous 21 days, into Row Cell counts.

(3) 10 mouse of control group are without any processing.

1.2 spleen tissue Foxp3⁺Treg and Th17 cell detections

1.3 statistical analysis

2 result testing results are as shown in table 11：

Table 11：

Cell	Control group	Model group	Treatment group
				Treg cell percentages	(0.50 ± 0.03) %	(0.40 ± 0.21) %**	(0.46 ± 0.25) %^Δ
Th17 cell percentages	(0.52 ± 0.02) %	(0.95 ± 0.17) %**	(0.82 ± 0.22) %^Δ
				Treg/Th17	0.96±0.03	0.42±0.20*	0.56±0.23^Δ

It is found according to model, after to animal model application D-MANNOSE, Treg cellular levels are improved, treatment group Clinical symptoms also improve to a certain extent.

Embodiment 11：Hyperthyroidism animal model experiment

1 materials and methods

The foundation of 1.1 animal packets and hyperthyroidism animal model

SD male rats, 350~390g of weight, purchased from Shandong University's Experimental Animal Center, rearing conditions with embodiment 1, Whole experiment process is carried out by animal protection and instruction policy.

(1) Hyperthyroid Model group：By levothyroxine sodium (L-T₄) 200 μ g (trade name Euthyrox, the pharmacy of Merck KGaA Lyons Group produces, and every contains 100 μ g) be dissolved in gavage in 3~5ml distilled water after grinding, once a day, heart rate after 21 days, blood pressure, The variation of the indexs such as cardiac weight, cardiac muscle cell's size, heart weight weight ratio and the variation base of the cardiovascular system of Patients with Hyperthyroidism This is consistent, display modeling success.

(3) 10 rats of control group give physiological saline.

1.2 spleen tissue Foxp3⁺Treg and Th17 cell detections

1.3 statistical analysis

2 result testing results are as shown in table 12：

Table 12：

Cell	Control group	Model group	Treatment group
				Treg cell percentages	(0.50 ± 0.03) %	(0.37 ± 0.21) %**	(0.42 ± 0.23) %^Δ
Th17 cell percentages	(0.55 ± 0.02) %	(0.90 ± 0.13) %**	(0.80 ± 0.32) %^Δ
				Treg/Th17	0.91±0.01	0.41±0.02*	0.53±0.26^Δ

Embodiment 12：Contact dermatitis animal model experiment

1 materials and methods

The foundation of 1.1 animal packets and contact dermatitis animal model

(1) model group：After dexamethasone tablet is first dissolved with alcohol, it is configured to the solution of 0.2mg/ml.It tests the 0th day small Mouse back shaving, 1d and 2d 1% (acetone of 5-isothiocyanate fluorescein (FITC)：Dibutyl phthalate=1：1) It is applied to mouse back shaving position and carries out sensitization, 7d FITC0.5% (acetone：Dibutyl phthalate=1：1) it is applied to The left ear excitation of mouse.

(2) treatment group：10 hello D-MANNOSE water (1.0M) of modeling mouse were freely drunk in the 3rd day from experiment, continuous 21 After it, Cell counts are carried out.

(3) 10 mouse of control group are without any processing.

1.2 spleen tissue Foxp3⁺Treg and Th17 cell detections

2 result testing results are as shown in table 13：

Table 13：

Cell	Control group	Model group	Treatment group
				Treg cell percentages	(0.45 ± 0.03) %	(0.38 ± 0.23) %**	(0.42 ± 0.24) %^Δ
Th17 cell percentages	(0.50 ± 0.02) %	(0.96 ± 0.17) %**	(0.72 ± 0.31) %^Δ
				Treg/Th17	0.90±0.03	0.40±0.22*	0.58±0.28^Δ

Embodiment 13：Eczema animal model experiment

1 materials and methods

The foundation of 1.1 animal packets and eczema animal model

(1) model group：Sensitization the previous day, back 3cm × 3cm regions are lost hair or feathers with depilatory cream, and next day hair removal section applies 5%2 outside, 50 μ L sensitization of 4- dinitrofluorobenzene (DNCB) the 1st time；After 2 weeks, back depilation 3cm × 3cm, next day, outer painting 0.1%2,4- dinitros 100 μ L of base chlorobenzene (DNCB) are excited, 1 times/week, continuous 4 weeks, through pathological section verification modeling success.

(3) 10 mouse of control group are without any processing.

1.2 spleen tissue Foxp3⁺Treg and Th17 cell detections

1.3 statistical analysis data are represented with mean ± standard deviation, using Graphad prism5.0 statistical softwares, two groups Between mean compare and examined using t, with P<0.05 is statistically significant for difference.、

2 result testing results are as shown in table 14：

Table 14：

Cell	Control group	Model group	Treatment group
				Treg cell percentages	(0.45 ± 0.03) %	(0.39 ± 0.23) %**	(0.42 ± 0.31) %^Δ
Th17 cell percentages	(0.50 ± 0.02) %	(0.90 ± 0.16) %**	(0.73 ± 0.20) %^Δ
				Treg/Th17	0.90±0.03	0.43±0.21*	0.57±0.24^Δ

Embodiment 14：Autoimmune uveitis animal model experiment

1 materials and methods

The foundation of 1.1 animal packets and Autoimmune uveitis (EAU) animal model

Lewis male rats, weight about 160~180g, purchased from Shandong University's Experimental Animal Center, rearing conditions are the same as implementation Example 1, whole experiment process are carried out by animal protection and instruction policy.

(1) model group：Retinoid binding protein between photoreceptor (IRBP, Shanghai life work) 30 μ g and PBS30 μ l are mixed It closes, then adds in mycobacterium tuberculosis (H37Ra, Difco companies of the U.S.) after 0.5mg, 30 μ l of Freund's complete adjuvant (CFA) are fully emulsified It is injected to Rats With Unilateral metapedes private school to be subcutaneously immunized, makes experimental autoimmune uveoretinitis (EAU) model；After immune 8 to 9 extremely big rathole prosthomeres have inflammation appearance, show as iris vessels expansion, and dyscoria is shunk, and anterior chamber is slight or moderate is mixed Pond；9 days experimental rat anterior ocular segment inflammation peak after immune, show as anterior chamber's severe and mix pond, pupil is visible or invisible, companion Or not with empyema.Verify modeling success.

(2) treatment group：10 hello D-MANNOSE water (1.0M) of modeling rat were freely drunk in the 3rd day from experiment, dripped simultaneously Eye (106.5mM NaCl, 26.1mM NaHCO₃、18.7mM KCl、1.0mM MgCl₂、0.5mM NaH₂PO₄、1.1mM CaCl₂ 10mM HEPE, 0.2M D-MANNOSEs), 3 times a day, 3~5 drip every time；After continuous 7 days, Cell counts are carried out.

(3) 20 rats of control group are without any processing.

1.2 spleen tissue Foxp3⁺Treg and Th17 cell detections

2 result testing results are as shown in Table 15：

Table 15：

Cell	Control group	Model group	Treatment group
				Treg cell percentages	(0.44 ± 0.03) %	(0.37 ± 0.16) %**	(0.40 ± 0.21) %^Δ
Th17 cell percentages	(0.52 ± 0.02) %	(0.91 ± 0.18) %**	(0.71 ± 0.25) %^Δ
				Treg/Th17	0.84±0.03	0.40±0.21*	0.56±0.24^Δ

Embodiment 15：Immune membranous conjunctivitis animal model experiment

1 materials and methods

The foundation of 1.1 animal packets and immune membranous conjunctivitis animal model

Cleaning grade Balb/c mouse are purchased from Shandong University's Experimental Animal Center, and rearing conditions were entirely tested with embodiment 1 Journey is carried out by animal protection and instruction policy.

(1) model group：It will delay on the day of sensitization containing 100 μ g PBS of sensibiligen chicken ovalbumin OVA (sigma companies of the U.S.) The mixture of the common 0.5ml of fliud flushing makes a group mouse sensitization through intraperitoneal injection.In experiment the 10th day, OVA (5mg/ml) is dissolved in Ph7.2 PBS liquid in instill the eyes of mouse and carry out sensibiligen attack and face eastern symptom with induce immune membranous conjunctivitis.Through pathological section Verify modeling success.

(2) treatment group：10 hello D-MANNOSE water (1.0M) of modeling mouse were freely drunk in the 3rd day from experiment, dripped simultaneously Eye (106.5mM NaCl, 26.1mM NaHCO₃、18.7mM KCl、1.0mM MgCl₂、0.5mM NaH₂PO₄、1.1mM CaCl₂ 10mM HEPE, 0.2M D-MANNOSEs), 3 times a day, 3~5 drip every time；After continuous 7 days, Cell counts are carried out.

(3) 20 mouse of control group are without any processing.

1.2 spleen tissue Foxp3⁺Treg and Th17 cell detections

2 results

Testing result is as shown in table 16：

Table 16：

Cell	Control group	Model group	Treatment group
				Treg cell percentages	(0.48 ± 0.02) %	(0.42 ± 0.16) %**	(0.45 ± 0.21) %^Δ
Th17 cell percentages	(0.64 ± 0.05) %	(0.92 ± 0.18) %**	(0.71 ± 0.35) %^Δ
				Treg/Th17	0.75±0.03	0.46±0.21*	0.63±0.28^Δ

Embodiment 16：Xerophthalmia animal model experiment

1 materials and methods

The foundation of 1.1 animal packets and xerophthalmia animal model

Using Adult female New Zealand White Rabbit, weight all New Zealand White Rabbit before 2.5~3kg, experiment receive clinic Eye examination excludes have eye disease animal.

Rabbit is randomly divided into control group, model group and treatment group (every group 10)：

(1) model group：Lachrymal gland is transferred to and fills after winning left side glandula lacrimalis inferior in animal super-clean bench by rabbit anesthesia In the centrifuge tube of 5mlHam ' s liquid, rabbit lacrimal epithelial cell is detached；Self lacrimal epithelial cell is transferred to 96 orifice plates and 24 orifice plates are adopted It is independently cultivated 2 days with the full culture mediums of DMEM, using 96 orifice plates (cell density 3*10⁶A/ml adds 100 μ l per hole) culture it is thin Born of the same parents are for BrdU kits detection proliferation；Using 24 orifice plates (cell density 1.8 × 10⁷A/ml, per 400 μ l of hole) culture it is thin Born of the same parents are used to be mixed.Gamma-rays irradiates, it is made to keep antigenicity.Rabbit peripheral blood lymphocytes is detached, the lymph of separation is thin Born of the same parents' recycling is put into centrifuge tube, is resuspended after centrifugation, adjusts cell density, prepares the cell of density corresponding with lachrymal gland cell respectively Liquid.96 orifice plates add in lymphocyte 3 × 10 per hole⁵A, orifice plate adds in lymphocyte 1.8 × 10 per hole⁶It is a.Mixing co-cultures 4 days Afterwards, BrdU methods (BrdU kits, roche Products) detection lymphopoiesis ratio is 3.71.Model group passes through ear edge The autologous leukocytes 5 × 10 of venous re-transfusion activation⁴.Compared with normal group, dry eye model group lacrimal secretion substantially reduces, tear film Rupture time shortens fluorescein sodium dyeing and shows that cornea diffuses dotted coloring.After being injected intravenously the lymphocyte week of activation, lachrymal gland Lymphocytic infiltration, display modeling success are shown with conjunctival tissue dyeing.

(2) treatment group：Modeling success 10 hello D-MANNOSE water (1.0M) of rabbit are freely drunk, after continuous 7 days, are dripped simultaneously Eye (106.5mM NaCl, 26.1mM NaHCO₃、18.7mM KCl、1.0mM MgCl₂、0.5mM NaH₂PO₄、1.1mM CaCl₂ 10mM HEPE, 0.2M D-MANNOSEs), 3 times a day, 3~5 drip every time；Carry out Cell counts.

(3) 10 rabbits of control group are without any processing.

1.2 spleen tissue Foxp3⁺Treg and Th17 cell detections

After intramuscular anesthesia, separating spleen.The spleen of taking-up is placed in the Tissue Culture Dish for filling RPMI-1640 culture mediums In, it mills.Pasteur pipet piping and druming is uniform, filtering, 2000rpm centrifugations 8min.Abandon supernatant, 20ml RPMI-1640 culture base weights Outstanding cell, is added on 20mlFicoll, centrifuges, 2000rpm, 20min.Draw intermediate misty buffy coat, 20ml RPMI-1640 culture mediums, 2000rpm, 20min are washed twice, and cell is resuspended in l RPMI-1640 culture mediums, are counted.Take 10⁵Carefully Born of the same parents add in buffer solution 30~50 μ l, FITC-antiRabbit CD4, PE-antihuman-FOXP3 and respectively add in 2 μ l dyeing, keep away Light is put into 4 degree of refrigerators, and solution is educated 60 minutes.Streaming pipe is taken out, 1500rpm, 10min is centrifuged, gently abandons supernatant.Add in buffer solution from The heart 1500rpm, 10min, this step of repetition is primary, and buffer solution is resuspended, upper FACS (flow cytometer, U.S. company BD) detection.

1.3 statistical analysis

2 result testing results are as shown in table 17：

Table 17：

Cell	Control group	Model group	Treatment group
				Treg cell percentages	(6.5 ± 0.03) %	(3.8 ± 0.23) %**	(5.2 ± 0.31) %^Δ

Embodiment 17：Allergic rhinitis animal model experiment

1 materials and methods

The foundation of 1.1 animal packets and allergic rhinitis animal model

(1) model group：Model group is aided with aluminum hydroxide adjuvant sensitization with ovalbumin, with 40% ovalbumin aluminium hydroxide PBS suspensions mouse peritoneal injects 200 μ L (containing ovalbumin 20mg), is immunized again with same procedure within the 14th day.Blank group is small PBS solution is injected intraperitoneally in the same time in mouse.It is 21st day immune, (contain ovalbumin 100 with 1% ovalbumin PBS solution, 20 μ L μ g) collunarium excitation, 6d is continuously repeated, establishes mouse allelgic rhinitis models.

(2) treatment group：10 hello D-MANNOSE water (1.0M) of modeling mouse were freely drunk in the 3rd day from experiment, continuous 14 After it, Cell counts are carried out.

(3) 20 mouse of control group are without any processing.

1.2 spleen tissue Foxp3⁺Treg and Th17 cell detections

1.3 statistical analysis

2 result testing results are as shown in table 18：

Table 18：

Cell	Control group	Model group	Treatment group
				Treg cell percentages	(0.54 ± 0.03) %	(0.32 ± 0.27) %**	(0.45 ± 0.31) %^Δ
Th17 cell percentages	(0.76 ± 0.02) %	(0.97 ± 0.19) %**	(0.86 ± 0.20) %^Δ
				Treg/Th17	0.71±0.03	0.33±0.24*	0.52±0.27^Δ

Embodiment 18：Irritable bowel syndrome animal model experiment

1 materials and methods

The foundation of 1.1 animal packets and irritable bowel syndrome animal model

Adult male SD rats 60,250~350g of weight, purchased from Shandong University's Experimental Animal Center, rearing conditions are same Embodiment 1, whole experiment process are carried out by animal protection and instruction policy.

(1) model group：Model group rats are given the physiological saline suspension containing 350~400 trichinella larvae cysts and are filled Stomach.Through through pathological section verification modeling success after 2 weeks.

(2) treatment group：10 hello D-MANNOSE water (1.0M) of modeling rat were freely drunk in the 2nd day from modeling, continuous 14 After it, Cell counts are carried out.

(3) control group gives normal saline gavage.

1.2 peripheral blood Foxp3+Treg cell detections

Rat vein blood 2ml is taken, using EDTA anti-freezings, using fluorescence direct labelling method and flow cytomery peripheral blood Mononuclearcell (PBMC) film surface CD4, CD25.Each 20 μ l of monoclonal antibody of different fluorescent markers are taken, are separately added into 100 μ l The peripheral blood of anticoagulant heparin is incubated 15min at room temperature, adds in erythrocyte cracked liquid, PBS is washed 2 times, with flow cytometer (BD Company, FACS Cali2bur) it detects, software kit analysis data record positive percentage, subtract nonspecific control value Peripheral blood Treg percentage test statistical analyses.

1.3 statistical analysis

2 result testing results are as shown in table 19：

Table 19：

Cell	Control group	Model group	Treatment group
				Treg cell percentages	(3.90 ± 0.15) %	(2.26 ± 0.13) %**	(3.34 ± 0.21) %^Δ

Embodiment 19：Cataract animal model is tested

1 materials and methods

The foundation of 1.1 animal packets and cataract animal model

7 day age not opening eyes, health, cleaning grade SD rats 60,20~22g of weight, conventional breast-feeding, purchased from mountain Eastern university's Experimental Animal Center, rearing conditions with embodiment 1, whole experiment process by animal protection and instruction policy into Row.

(1) model group：Asia is subcutaneously injected by 3.46mg/kg weight (i.e. 20 μm of ol/kg weight) neck to 10 day age rat Sodium selenate, the next day primary, co-injection 3 times.For the first time in injection sodium selenite 30 minutes, physiology is injected intraperitoneally by 0.1mg/10g bodies Brine, co-injection 6 days.Modeling success is shown through slit lamp microscope observation.

(2) treatment group：10 hello D-MANNOSE water (1.0M) of modeling rat were freely drunk in the 2nd day from modeling, dripped simultaneously Eye (106.5mM NaCl, 26.1mM NaHCO₃、18.7mM KCl、1.0mM MgCl₂、0.5mM NaH₂PO₄、1.1mM CaCl₂ 10mM HEPE, 0.2M D-MANNOSEs), 3 times a day, 3~5 drip every time；After continuous 14 days, Cell counts are carried out.

(3) control group gives normal saline gavage.

1.2 spleen tissue Foxp3⁺Treg and Th17 cell detections

2 result testing results are as shown in table 20：

Table 20：

Cell	Control group	Model group	Treatment group
				Treg cell percentages	(0.50 ± 0.03) %	(0.41 ± 0.24) %**	(0.46 ± 0.22) %^Δ
Th17 cell percentages	(0.65 ± 0.02) %	(0.77 ± 0.32) %**	(0.72 ± 0.32) %^Δ
				Treg/Th17	0.77±0.03	0.53±0.24*	0.63±0.27^Δ

Experimental example 1

Leucoderma (vitiligo) is a kind of common multiple depigmentation dermatoses, the definite pathogenesis of leucoderma It not yet illustrates completely, there are many hypothesis at present.Wherein theory of autoimmunity receives more and more attention.Most scholar's researchs are recognized For patients with vitiligo compared with normal person CD4⁺CD25⁺Treg cells reduce, especially general hair property patients with vitiligo and normal person's phase Compare CD4⁺CD25⁺Treg cells significantly reduce.

This experimental example uses CD4 in Flow cytometry patients with vitiligo peripheral blood⁺CD25⁺The quantity of Treg cells, and With RT-polymerase chain reaction (RT-PCR) method to pretherapy and post-treatment patients with vitiligo CD4⁺CD25⁺In Treg cells The expression of Foxp3mRNA is detected.

1 material and method

1.1 Specimen origin：24 patients with vitiligo are Dermatology Outpatient Department patient, meet national pigment disease group 2003 The effect of December in year formulates criterion.

It fully recovers and all subsides for hickie, restore normal skin tone；Effective is hickie partial remission or diminution, restores normal skin tone Area account for skin lesion area >=50%；It improves as hickie partial remission or diminution；Expand in vain for the regeneration of hickie non-pigment or range Greatly；Effectively=recovery from illness is+effective+to improve；Effective=recovery from illness+effective.Using the peripheral blood of 16 normal volunteers as normal control. Patient and family members, volunteer are to testing informed consent.

Oral medication, 40g/ natural gift are taken for 2 times, 28 days as a treatment course.

1.2 instruments and reagent：Flow cytometer is U.S. company BD product (model：FACSCalibur).The mouse of PE labels Anti-human CD4 (IgG-PE), the anti-human CD25 of mouse (IgG-PE) and 1/ γ 2 of Isotype control γ of FITC labels are purchased from U.S. company BD. Ficoll separating liquids, TRIZOL, Ex-Taq archaeal dna polymerase, dNTP, MML-V reverse transcriptase, M2MLV reverse transcriptases etc. are purchased from crystalline substance Biotech firm of U.S., primer are synthesized by Shanghai Sheng Gong biotech firms.

1.3 method

1.3.1 peripheral blood CD4⁺CD25⁺The detection of Treg cells：All samples are taken from early morning limosis vein blood, and heparin resists It is solidifying.100 μ L whole bloods are taken, are separately added into each 10 μ l of anti-CD4, anti-CD 25 antibody, separately set control tube, add in homotype negative control γ 1/ γ 2 is incubated at room temperature 20~30min, adds in erythrocyte cracked liquid, vibrates mixing, is incubated at room temperature 10min.2ml PBS are with 1000r/ Min washs 5min, abandons supernatant, cell is suspended in 0.5ml PBS cleaning solutions, and upper machine testing lymphocyte is sub- after vibrating mixing Group and CD4+CD25+Treg cell subsets are horizontal.

1.3.2 the separation of peripheral blood mononuclear cells (MNCS)：Patient and collator anticoagulant heparin 3~5ml of peripheral blood are taken, PBS dilutes, and lymphocyte separation medium (Ficoll) isolates mononuclearcell, and PBS is washed 2 times, with containing 10% calf serum RPMI-1640 will adjust cell to 1 × 10⁹/L。

1.3.3 the synthesis of Total RNAs extraction and cDNA：By TRIzol (Shanghai Sangon Biotech Company's product) reagent manufacture specifications into Row MNCs Total RNAs extractions, agarose gel electrophoresis identify the quality of RNA, and absorbance scanning calculates total rna concentration, A260/A280 Ratio is 1.8~2.0.Take 5 162 μ l, 2.5mmol/L dNTP of μ g, 50pmol/LOligo (dT) of cell total rna 3 μ l, DEPC The distilled water of processing is supplemented to 34 μ l, is put into ice water and centrifuges after 65 DEG C of 5min, then sequentially adds M2MLV5 × reverse At 10 μ l, 0.1mmol/L DDT of record enzyme buffer liquid 4 μ l, M2MLV reverse transcriptases 200U (Invitrogen Products), DEPC The distilled water of reason is supplemented to stopped reaction after 50 μ l, 37 DEG C of 60min, 70 DEG C of 15min.Gained cDNA is in -20 DEG C of preservations.

1.3.4RT-PCR detect Foxp3mRNA expressions：It is public by Shanghai life work with reference to document [9] design primer after text Department's synthesis, is dissolved as 20 μm of ol/L, -20 DEG C save backup with the sterilizing distilled water of no RNA enzyme.

Foxp3 primer sequences are as follows：

Upstream：5'-AGG CTT CAT CT G TGG CAT CAT-3', (SEQ ID NO:1)；

Downstream：5'-CTT GCGGAA CTC CAG CTC AT-3', (SEQ ID NO:2)；

Expected product length 443bp；

Internal reference β-actin sequences are as follows：

Upstream：5'-ATG GAT GAT GAT ATC GCG CG-3', (SEQ ID NO:3)；

Downstream：5'-CATGAA GCA TTTGCG GTG GAC GAT GGA GGGGCC-3', (SEQ ID NO:4)；

Expected product length 1126bp.

Reaction condition is 94 DEG C of denaturation 40s, 60 DEG C of annealing 40s, 72 DEG C of extension 60s, 35 cycles.2% agarose of product Electrophoresis, gel imaging system scanning.Foxp3mRNA expressions are expressed with Foxp3/ β-actin.

1.4 statistical method：Data analysis is carried out using 10.0 softwares of SPSS, data are represented with x ± s, group difference ratio Compared with two independent samples t tests are used, with comparing before and after treatment between group using paired t-test, P<0.05 has statistics meaning for difference Justice.

2 Results Test results are as shown in table 21~23.

Table 21：Patient and CD4 in Normal group peripheral blood⁺CD25⁺The comparison of Treg cell quantities

(x ± s, %)

Group	Number of cases	CD4+T	CD4⁺CD25⁺T	CD4⁺CD25⁺T/CD4⁺T
					Healthy control group	16	39.32±8.21	7.89±2.87	6.8±3.87
Patient group	24	25.54±7.32	2.87±3.54*	2.3±2.76*

*P<0.05。

Table 22：CD4 in pretherapy and post-treatment peripheral blood⁺CD25⁺The expression water of Treg cell quantities and Foxp3mRNA

Flat variation (x ± s)

Group	Number of cases	CD4⁺CD25⁺T (%)	Foxp3/β-actin(A)
				Before treatment	24	2.67±3.53	0.25±0.15
After treatment	24	5.54±4.12*	0.75±0.32*

*P<0.05。

Table 23：The variation (x ± s) of pretherapy and post-treatment hickie part

Group	Number of cases	It is efficient
			Patient group	24	58%

As a result it shows：Patient CD4 after treatment⁺CD25⁺The quantity of Treg cells significantly increases (P compared with pre-treatment< 0.05), the expression of Foxp3mRNA has significantly raising (P in cell before relatively treating<0.05).Illustrate by effectively treatment, with CD4⁺CD25⁺The raising of the expression of Foxp3mRNA in Treg cells, patients with vitiligo CD4⁺CD25⁺Treg cells are exempted from Epidemic disease regulatory function is restored, and autoreactive T cell activity and function is inhibited to strengthen, the state of an illness of patient is made to be eased.

2 Behcet's disease of experimental example

1 material and method

1.1 research object

The Behcet's disease patient totally 44 that in January, 2013~2015 year have been clarified a diagnosis May, wherein female 38, man 6, 18~52 years old age.All meet the diagnostic criteria that the international Behcet's disease discussion of nineteen ninety-five the 5th proposes.Wherein clinical table Existing recurrent oral ulceration (3 times/year of ＞) person 44, genitals and ulcus vulvae 36, erythema nodosum 27, arthralgia 19, eye iris cyclitis or conjunctivitis 20.By patient assessment's sequence, it is randomly divided into two groups, every group 22.Two groups of patients Gender, age, the course of disease and skin lesion etc. difference are not statistically significant (P ＞ 0.05), are comparable.All patients not companion There is whole body system disease, do not received immune modulating treatment, and without past medical history and family history, whole voluntary participations before medical This research.

1.2 research method

Patient gives D-MANNOSE 40g/ natural gift and takes for 2 times, and 8 weeks as a treatment course.Be discontinued after 8 weeks, be discontinued 3 months it is laggard Row curative effect is judged.

1.3 observation curative effect index

Fully recover and completely disappeared for clinical symptoms and sign, and laboratory examination is without exception, be discontinued 3 months in it is asymptomatic multiple Hair；It effectively disappears or mitigates for clinical symptoms and sign, laboratory examination is largely normal, but it is Symptomatic in 3 months to be discontinued Recurrence；It is not improved in vain for clinical symptoms and sign, laboratory examination is all abnormal.In terms of cure rate and efficient addition It calculates efficient.

2 result experimental results are as shown in table 24.

Table 24：

Group	Number of cases	It is efficient
			Patient group	24	56%

3 glaucoma of experimental example

1 material and method

1.1 research object

In August, 2014 in August, -2016 is diagnosed as POAG patient 49 (90), meets Chinese Medical Association blueness in 1987 The diagnosis of glaucoma standard that light eye group is formulated.26~68 years old age, the median age 53 years old, the course of disease are 3 months~5 years (middle positions Number 2 years), eyesight is 0.4~1.0.

1.2 research method

It is randomly divided into two groups, experimental group patient gives D-MANNOSE 40g/ natural gift and takes, while eye drip (106.5mM for 2 times NaCl、26.1mM NaHCO₃、18.7mM KCl、1.0mM MgCl₂、0.5mM NaH₂PO₄、1.1mM CaCl₂ 10mM HEPE、 0.2M D-MANNOSEs), 3 times a day, 3~5 drip every time；Curative effect judge is carried out after 8 weeks.

1.3 observation curative effect index:10:00 or 14:00 single intraocular pressure is effective no more than 15mmHg.

2 result experimental results are as shown in Table 25.

Table 25：

Group	Number of cases	It is efficient
			Patient group	90	65%

It is not for limiting claim, any this field skill although the application is disclosed as above with preferred embodiment Art personnel can make several possible variations and modification under the premise of the application design is not departed from, therefore the application Protection domain should be subject to the range that the application claim is defined.

<110>It is noble first

<120>Mannose is for improving the new application of Treg cell quantities and its Foxp3 factor expressions level

<160>4

<210>1

<211>21

<212> DNA

<213>Artificial sequence

<400>1

AGGCTTCATCTGTGGCATCAT

<210>2

<211>20

<212> DNA

<213>Artificial sequence

<400>2

CTTGCGGAACTCCAGCTCAT

<210>3

<211>18

<212> DNA

<213>Artificial sequence

<400>3

GGATGATGATATCGCGCG

<210>4

<211>33

<212> DNA

<213>Artificial sequence

<400>4

CATGAAGCATTTGCGGTGGACGATGGAGGGGCC

Claims

1. application of the mannose in the drug for the treatment of autoimmune disease, food, health products or cosmetics are used to prepare.

2. application according to claim 1, which is characterized in that the autoimmune disease is due to Treg cell quantities Autoimmune disease caused by reduction.

3. application according to claim 1, which is characterized in that the autoimmune disease is selected from lupus erythematosus, first shape It is gland hyperfunction, I types or type-2 diabetes mellitus and its complication, IgA ephritis, mouth xerophthalmia scheorma syndrome, rheumatoid arthritis, strong Straightforward rachitis, simple obesity, bronchial asthma, neurodermatitis, canker sore, ulcerative colitis, ox-hide moss, leucoderma Wind, autoimmune eczema, Autoimmune uveitis, autoimmune eye iris cyclitis, itself is exempted from Behcet's disease Epidemic disease membranous conjunctivitis, autoimmune xerophthalmia, autoimmune cataract, autoimmune glaucoma, allergic rhinitis, intestines are easy Bowel syndrome, pruitus.

4. application of the mannose in drug, food, health products or the cosmetics for safeguarding immune homeostasis are used to prepare.

5. application according to claim 4, which is characterized in that the disease as caused by the immune system homeostasis is selected from Lupus erythematosus, hyperthyroidism, I types or type-2 diabetes mellitus and its complication, IgA ephritis, mouth xerophthalmia scheorma syndrome, class wind Wet arthritis, ankylosing spondylitis, simple obesity, bronchial asthma, neurodermatitis, canker sore, ulcerative colitis Inflammation, ox-hide moss, leucoderma, Behcet's disease, autoimmune eczema Autoimmune uveitis, autoimmune eye iris eyelash Shape body inflammation, autoimmunity membranous conjunctivitis, autoimmune xerophthalmia, autoimmunity sexual factor cataract, autoimmune green light Eye, allergic rhinitis, irritable bowel syndrome, pruitus.

6. mannose improves drug, food, the health products of Treg cell quantities and its Foxp3 factor expressions level being used to prepare Or the application in cosmetics.

7. according to the application described in claim 1~6 any claim, the mannose is D-MANNOSE.