CN105288617B - A kind of method and drug inhibiting Echinococcus granulosus - Google Patents
A kind of method and drug inhibiting Echinococcus granulosus Download PDFInfo
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- CN105288617B CN105288617B CN201510689071.1A CN201510689071A CN105288617B CN 105288617 B CN105288617 B CN 105288617B CN 201510689071 A CN201510689071 A CN 201510689071A CN 105288617 B CN105288617 B CN 105288617B
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- echinococcus granulosus
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Abstract
The invention discloses a kind of methods inhibiting Echinococcus granulosus, by using the infection of 3 antibody blocking Echinococcus Granulosus Cysts of anti-Tim.Technical solution disclosed in this invention is directly enhanced by the Echinococcus granulosus object macrophage phagocytic function of itself by using 3 antibody of anti-Tim, to improve its immunocompetence to Echinococcus Granulosus Cysts.
Description
Technical field
The present invention relates to a kind of methods inhibiting Echinococcus granulosus and treatment since Echinococcus granulosus causes to wrap
The drug of parasitosis belongs to biology and field of medicaments.
Background technology
Echinococcosis is a kind of metacestode parasitized larvae by echinococcus (Echinococcus granulosus) in human body
A kind of infecting both domestic animals and human parasitic disease seriously endangering health caused by the internal organs such as liver, lung, in China, such disease includes mainly capsule
Two kinds of type echinococcosis (CE) and alveolar echinococcosis (Alveolar echinococcosis, AE), respectively by Echinococcus granulosus children
Worm (echinococcus) and Echinococcus multilocularis larva (Echinococcus multilocularis) colonize in caused by the histoorgan of human body.Among both,
Cystic echinococcosis, also known as echinococcosis granulosa can form cycle between animal and animal or between humans and animals, become nature
Epidemic disease source property disease, is the main species at present there is an urgent need for prevention.
Currently, there is no good prophylactic treatment scheme for the treatment of echinococcosis.It is typically clinically to be controlled using operation
It treats.In clinical practice, lead to Echinococcus hydatid cyst heeling-in because cyst fluid is excessive when the shortcoming of surgical operation therapy is operation；Meanwhile
It performs the operation the smaller hydatidoma in difficult liver parenchyma depths, particularly with case old and frail or with important organ illness,
And for Multiorganic hydatidosis and involve multiple internal organs persons, surgical operation just seems particularly troublesome, especially because Bile fistula
Secondary abdominal echinococcosis of tumour rupture when can perform the operation caused by cyst fluid extravasation plantation, even if Repeated Operation, difficult all extract；
For more house type hepatic echinococcosis that lesion infiltrates extensively, operation is even more helpless.
In recent years, the chemotherapy and immunization therapy of echinococcosis are being studied always both at home and abroad, chemotherapy is faced with toxicity
Larger, narrow application range defect, and immunization therapy does not obtain more significant achievement always for many years, such research focuses mostly on
In immunodiagnosis.Due to the missing of therapy, this field is equally faced with above-mentioned serious in the treatment of animal echinococcosis
The problem of.The especially existing therapeutic scheme cost for Human Hydatidosis is higher, and nothing is answered in the treatment of animal echinococcosis
With value.
Invention content
In view of the deficiencies of the prior art, applicant causes the biological process of echinococcosis to carry out Echinococcus granulosus
Further investigation, especially carefully analyzes and has probed into the interactively between Echinococcus granulosus and immunologic process, to send out
A kind of method inhibiting Echinococcus granulosus is illustrated, and can be made into treatment Echinococcus granulosus based on this to lead to echinococcosis
Drug.
Specifically, the present invention is achieved through the following technical solutions：
A method of inhibiting Echinococcus granulosus, uses the infection of anti-Tim-3 antibody blockings Echinococcus Granulosus Cysts.
The experiment that applicant carries out is shown, by using anti-Tim-3 antibody, can be reduced the expression of Tim-3, be improved macrophage
The phagocytic function of cell enhances the immune killing to Echinococcus Granulosus Cysts, reduces its immunologic escape.
Preferably, relative to 1000 Echinococcus granulosus amounts, the dosage of anti-Tim-3 antibody is 30-80ug.
It should be readily apparent to one skilled in the art that coming into force for the ease of anti-Tim-3 antibody, when using anti-Tim-3 antibody also
It can synchronize using anti-Fc antibody, inhibit combinations of the Tim-3 to macrophage, improve the effectiveness of anti-Tim-3 antibody.
When using anti-Fc antibody, dosage is identical as anti-Tim-3 antibody dosage can to meet needs；However this not must
Must, those skilled in the art can rationally adjust the dosage of anti-Fc antibody according to actual needs.
Based on above-mentioned achievement, the invention also discloses a kind of medicines for treating echinococcosis caused by Echinococcus granulosus
Object, including anti-Tim-3 antibody is as active constituent.
Similarly, in the preferred case, anti-Fc antibody identical with anti-Tim-3 antibody dosage is also may include in said medicine.
Specific implementation mode
In order to illustrate the effect of technical solution of the present invention, applicant tests by taking specific scientific research and testing as an example.Ability
Field technique personnel it should be understood that technical solution as disclosed below is only a kind of verification and the explanation to the principle of the invention, and
The details of technical solution of the present invention, such as various operating process, dosage, application mode are not limited.
In following experiments, pathogen used be Echinococcus Granulosus Cysts, acquired, cultivate after vigor>90% for testing；
Mouse used be female BAl BIc/c mouse, 6-8 weeks, 20 ± 2g of weight.
In following, associated antibodies used can be bought from Reagent Company, such as the sale of Ebioscience companies is anti-
Similar antibody class commodity are also sold on the market by Fc antibody, anti-Tim-3 antibody, anti-igg antibody, other antibody companies.
Initially set up Echinococcus granulosus mouse model：It is random to divide by 24 healthy female BAl BIc/c mouse
At experimental group and control group.Infected group：A concentration of 10000/ml protoscolexs of intraperitoneal inoculation, 0.2ml/ is only；Control group：Abdominal cavity
It is inoculated with PBS, 0.2ml/ only, put to death mouse, carry abdominal cavity respectively by aseptic collection peripheral blood when 1,5,9,13 day, vertebra dislocation method
Macrophage, and spleen is won, prepare splenocyte suspension.
Next establishes Echinococcus granulosus mouse model after Tim-3 is blocked：By 24 healthy female BAl BIc/c
Mouse is randomly divided into experimental group and blocking group.Infected group：The anti-igg antibody of intraperitoneal inoculation 200ug；Blocking group：It is inoculated with 100ug
Anti- Fc antibody and the anti-Tim-3 antibody of 100ug, second day, a concentration of 10000/ml protoscolexs of each group intraperitoneal inoculation, 0.2ml/
Only, respectively after infection 1,5,9,13 day when aseptic collection peripheral blood, vertebra dislocation method put to death mouse, it is thin to put forward abdominal cavity macrophage
Born of the same parents, and spleen is won, prepare splenocyte suspension.
After the completion of above-mentioned, Isolation of Macrophages From Mouse Peritoneal Exudate is acquired according to biological method, and carry out macrophage and gulp down
Bite Function detection each inflammation-associated cytokine content related to macrophage.
In Echinococcus granulosus mouse model, experimental result is shown：
Flow cytometry Granulosus Cyst In Mice infects the expression (table 1) of Tim-3 on early stage splenic macrophage, carefully
Grain echinococcus early infection mouse after, the 1st, 5 day after infection, infected group (9.0 ± 2.14%~8.03 ± 1.17%) with compare
The expression of Tim-3 is not significantly changed on group (6.1 ± 1.2%~7.5 ± 0.8%) splenic macrophage, through statistical analysis, no system
Meter learns difference (P>0.05).9,13 days after infection, infected group (11.63 ± 0.50%~12.37 ± 0.21%) and control group
The expression of Tim-3 is all increased on (7.1 ± 0.9%~7.8 ± 1.3%) splenic macrophage.
The expression of Tim-3 on 1 Echinococcus Granulosus Cysts early infection mouse different time splenic macrophage of table
Note：Infected group compared with the control group, *：P<0.05.
Flow cytometry Granulosus Cyst In Mice infects the expression (table 2) of Tim-3 on early stage peritoneal macrophage,
Except the 1st day after infection, on the 5th, 9,13 day peritoneal macrophage the expression of Tim-3 all increase, through statistical analysis, infected group with
The expression raising of control group, Tim-3 has significant difference (P<0.05).
The expression of Tim-3 on 2 Echinococcus Granulosus Cysts early infection mouse different time peritoneal macrophage of table
Note：Infected group compared with the control group, *：P<0.05.
It is thin relative to control group macrophage related inflammatory that Granulosus Cyst In Mice infection early infection group is detected by QRT
The mRNA expressions of intracellular cytokine, are shown in Table 3.
The mRNA expressions of each inflammatory factor after 3 mouse infection Echinococcus Granulosus Cysts of table
Note：Infected group compared with the control group, *：P<0.05.
Upper table shows that TNF-α expresses raising after infection, and IL-6 expresses raising after infection, and IL-10 is expressed after infection
It increases, IL-1 β express raising after infection, and only expression in the 13rd day increases TLR2 after infection, TLR4 only the 1st day after infection
Expression increases, and only expression in the 13rd day increases HMGB1 after infection.
ELISA detects Granulosus Cyst In Mice and infects early infection group relative to control group macrophage related inflammatory cells
The protein expression (table 4) of the factor：
4 Echinococcus Granulosus Cysts early infection mouse different time macrophage associated inflammatory cytokine protein level table of table
It reaches
Note：Infected group compared with the control group, *：P<0.05.
From upper table, it can be seen that, TNF-α expresses raising after infection, and IL-6 expresses raising after infection, and IL-10 is feeling
It expresses and increases after dye, IL-1 β express raising after infection.
Chicken red blood cell is swallowed in vitro by Turnover of Mouse Peritoneal Macrophages, is observed Granulosus Cyst In Mice infection early stage respectively and is felt
Dye group and control group Peritoneal macrophage function and peritoneal macrophage phagocytic index (table 5) react the phagocytosis of macrophage
Function.
5 Echinococcus Granulosus Cysts early infection mouse different time macrophage phagocytic function of table
Note：Infected group compared with the control group, *：P<0.05.
Above-mentioned experimental result is shown, after Echinococcus Granulosus Cysts early infection mouse, comprehensive phagocytic rate and phagocytic index are as a result, huge
The phagocytic function of phagocyte reduces after infection.
Turnover of Mouse Peritoneal Macrophages NO burst sizes (table 6), Echinococcus Granulosus Cysts early infection are detected by Greiss reagent methods
After mouse, NO burst sizes increase.
The NO of 6 Echinococcus Granulosus Cysts early infection mouse different time macrophage of table discharges
Note：Infected group compared with the control group, *：P<0.05.
After Tim-3 blockings in Echinococcus granulosus mouse model, experimental result is shown：
Granulosus Cyst In Mice, which is detected, by QRT infects earlier T im-3 blockings group relative to infected group macrophage correlation
The mRNA expressions (table 7) of inflammatory cytokine：
After 7 anti-Tim-3 MAbs blockings of table after mouse infection Echinococcus Granulosus Cysts each inflammatory factor mRNA expressions
Note：Tim-3Ab+ infected groups are compared with infected group, *：P<0.05.
The above results are shown, are infected again and common infection Echinococcus Granulosus Cysts early infection mouse, TNF-α after blocking Tim-3
Further increase.
It is thin relative to infected group macrophage that Granulosus Cyst In Mice infection earlier T im-3 antibody blockings group is detected by ELISA
The protein expression level (table 8) of born of the same parents' associated inflammatory cytokine：
Each inflammatory factor albumen of Echinococcus Granulosus Cysts early infection mouse different time after the processing of 8 anti-Tim-3 MAbs blockings of table
Horizontal expression
Note：Tim-3Ab+ infected groups are compared with infected group, *：P<0.05.
The above results also show that with anti-Tim-3 MAbs blockings handle after Echinococcus Granulosus Cysts early infection mouse difference when
Between each inflammatory factor protein expression, TNF-α expression increases apparent.
Chicken red blood cell is swallowed in vitro by Turnover of Mouse Peritoneal Macrophages, is observed Granulosus Cyst In Mice infection early stage respectively and is felt
Dye group and Tim-3 blocking group Peritoneal macrophage functions and peritoneal macrophage phagocytic index (table 9) react macrophage
Phagocytic function, show Echinococcus Granulosus Cysts early infection mouse after, the phagocytic function of macrophage increases after infection.
Echinococcus Granulosus Cysts early infection mouse different time macrophage swallows work(after the processing of 9 anti-Tim-3 MAbs blockings of table
Energy
Note：Tim-3Ab+ infected groups are compared with infected group, *：P<0.05.
Turnover of Mouse Peritoneal Macrophages NO burst sizes (table 10), Echinococcus Granulosus Cysts early infection are detected by Greiss reagent methods
After mouse, Turnover of Mouse Peritoneal Macrophages NO burst sizes reduce after infection：
The NO releases of Echinococcus Granulosus Cysts early infection mouse different time macrophage after 10 Tim-3 of table is blocked
Note：Tim-3Ab+ infected groups are compared with infected group, *：P<0.05.
In summary experimental result can enhance Echinococcus Granulosus Cysts early infection it will be seen that by blocking Tim-3
Macrophage macrophage phagocytic function after mouse improves it to Echinococcus Granulosus Cysts, effectively reduces the immunologic escape of polypide, to
Improve the control effect to echinococcosis.
Claims (1)
1. a kind of anti-Tim-3 antibody exists in the application for the drug for preparing echinococcosis caused by treating Echinococcus granulosus, feature
In the infection using anti-Tim-3 antibody blockings Echinococcus Granulosus Cysts；
Relative to 1000 Echinococcus granulosus amounts, the dosage of anti-Tim-3 antibody is 30-80 μ g；
The anti-Tim-3 antibody is in preparing the application of drug of echinococcosis caused by treating Echinococcus granulosus, for treating
The drug of echinococcosis caused by Echinococcus granulosus, including anti-Tim-3 antibody is as active constituent.
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