JP7178041B2 - Hematopoietic progenitor cell activator and related technology - Google Patents

Description

TECHNICAL FIELD The present invention relates to a hematopoietic progenitor cell activator and a hematopoietic injury recovery agent useful for recovery from hematopoietic injury caused by radiation or the like.

In Medicine and Biology, 2012, Vol.156 No.10, pp.696-702, when chlorella powder was administered to mice that had been whole-body irradiated with 5 Gy of X-rays, platelets, leukocytes, granulocytes, prophase Erythroid progenitor cells (BFUE) and granulocyte-macrophage colony-forming cells (CFU-GM) increased with statistical significance compared to the saline-administered control group, but erythrocytes, lymphocytes, monocytes, It is shown that the increase or decrease in erythropoietin-dependent late erythroblastic progenitor cells (CFU-E) was not a significant (P < 0.05) variation.

Japanese Patent Application Laid-Open No. 2015-78160 discloses a radiation hematopoietic injury recovery agent containing a mixture of a cell wall crushed product of fruiting bodies of Agaricus blazei muril and a mycelium extract and a hot water extract of a cell wall crushed product of Chlorella. disclosed.

However, substances that can be obtained from chlorella and are useful for activating hematopoietic progenitor cells and for recovering hematopoietic disorders caused by radiation have not yet been identified.

Medicine and Biology, 2012, Vol.156 No.10, pp.696-702 JP 2015-78160 A

An object of the present invention is to provide a hematopoietic progenitor cell activator containing, as an active ingredient, a substance useful for the activation of hematopoietic progenitor cells obtained from chlorella and for the recovery of hematopoietic disorders caused by radiation and the like, and for the recovery of hematopoietic disorders caused by radiation. An object of the present invention is to provide a useful hematopoietic disorder recovery agent.

The hematopoietic progenitor cell activator and hematopoietic disorder recovery agent of the present invention can be expressed as follows.

(1) A hematopoietic progenitor cell activator comprising a peptide heteroglycan having the following physicochemical properties (a), (b), (c), (d) and (e) .
(a) average molecular weight: 1 million
(b) Specific rotation: [α] _D -11.6 (Measurement temperature 25°C)
(c) nitrogen content 5.39%, protein content 29.5%, polysaccharide content 70.3%;
(d) Sugar composition (molar ratio): Gal:GIc:Man:Xyl:Rha:Ara:Fru=32:25:13:8:7:5:3
(e) Amino acid composition (mol%):
Glu 15.1
Asp 13.8
Ala 9.9
Leu 8.9
Thr 6.5
Lys 6.2
Va1 5.9
Gly 5.8
Pro 5.5
Ser 5.4
Arg 4.8
Phe 3.6
Ileu 3.1
Tyr 2.1
His 1.7
Met 1.6
Cys 0.1
Total 100%

(2) The hematopoietic progenitor cell activation according to (1) above, wherein the hematopoietic progenitor cells are erythroblastic progenitor cells, erythropoietin-dependent monocytic progenitor cells, and neutrophil-granulocyte progenitor cells. agent.

(3) The hematopoietic progenitor cell activator according to (1) or (2) above, wherein the peptide heteroglycan is derived from a hot water extract of cell wall fragment of Chlorella pyrenoidosa.

(4) A hematopoietic disorder recovery agent comprising a peptide heteroglycan having the following physicochemical properties (a), (b), (c), (d) and (e) .
(a) average molecular weight: 1 million
(b) Specific rotation: [α] _D -11.6 (Measurement temperature 25°C)
(c) nitrogen content 5.39%, protein content 29.5%, polysaccharide content 70.3%;
(d) Sugar composition (molar ratio): Gal:GIc:Man:Xyl:Rha:Ara:Fru=32:25:13:8:7:5:3
(e) Amino acid composition (mol%):
Glu 15.1
Asp 13.8
Ala 9.9
Leu 8.9
Thr 6.5
Lys 6.2
Va1 5.9
Gly 5.8
Pro 5.5
Ser 5.4
Arg 4.8
Phe 3.6
Ileu 3.1
Tyr 2.1
His 1.7
Met 1.6
Cys 0.1
Total 100%

(5) recovery from hematopoietic disorder is achieved by increasing the number of red blood cells, platelets, white blood cells, granulocytes, lymphocytes and monocytes in peripheral blood, increasing spleen weight, recovery of splenic hematopoietic foci, increasing thymus weight, and The hematopoietic disorder recovery agent according to (4) above, which is manifested in recovery of thymic lymphocyte count.

(6) The hematopoietic disorder recovery agent according to (4) or (5) above, wherein the hematopoietic disorder is caused by irradiation.

(7) The hematopoietic disorder recovery agent according to any one of (4) to (6) above, wherein the peptide heteroglycan is derived from a hot water extract of cell wall fragment of Chlorella pyrenoidosa.

The hematopoietic progenitor cell activator and hematopoietic injury recovery agent of the present invention are effective in activating hematopoietic progenitor cells and in recovering from hematopoietic injury caused by radiation or the like, respectively.

Fig. 3 is a micrograph of a tissue section (hematoxylin-eosin staining) of the thymus of the 5.0 Gy whole-body irradiation group. 5. Photomicrographs of tissue sections (hematoxylin-eosin staining) of thymus in OGy whole-body irradiation/peptide heteroglycan-administered groups. Fig. 4 is a micrograph of a tissue section (hematoxylin-eosin staining) of the spleen of the 5.0 Gy whole-body irradiation group. 5. Photomicrographs of spleen tissue sections (hematoxylin-eosin staining) of OGy whole-body irradiation/peptide heteroglycan-administered groups.

(1) The hematopoietic progenitor cell activator and hematopoietic disorder recovery agent of the present invention comprise a peptide heteroglycan having the following physicochemical properties (a), (b), (c), (d) and (e) .
(a) average molecular weight: 1 million
(b) Specific rotation: [α] _D -11.6 (Measurement temperature 25°C)
(c) nitrogen content 5.39%, protein content 29.5%, polysaccharide content 70.3%;
(d) Sugar composition (molar ratio): Gal:GIc:Man:Xyl:Rha:Ara:Fru=32:25:13:8:7:5:3
(e) Amino acid composition (mol%):
Glu 15.1
Asp 13.8
Ala 9.9
Leu 8.9
Thr 6.5
Lys 6.2
Va1 5.9
Gly 5.8
Pro 5.5
Ser 5.4
Arg 4.8
Phe 3.6
Ileu 3.1
Tyr 2.1
His 1.7
Met 1.6
Cys 0.1
Total 100%

(2) The peptide heteroglycan of the present invention can be produced, for example, as follows.

Hot water extraction (for example, extraction with hot water at 95-100 ° C. Extraction time is, for example, 2 hours). However, it is not limited to this.), and the extract is obtained, for example, as a supernatant by standing still after extraction.

Low-molecular-weight substances with a molecular weight of 10,000 or less are removed from the resulting extract (preferably the supernatant obtained by centrifugation) by, for example, diafiltration or ultrafiltration to remove low-molecular-weight substances with a molecular weight of 10,000 or less. get the liquid

Absolute ethanol is added to this solution (for example, in a volume of 3 times) to obtain crude polysaccharide (FA) as a precipitate (for example, a precipitate after centrifugation). This crude polysaccharide (FA) is preferably obtained by washing the precipitate with absolute ethanol, then with anhydrous ether, and drying (preferably vacuum drying).

The resulting crude polysaccharide (FA) is dissolved in water and subjected to ion-exchange chromatography [for example, DEAE-Cellulose (Cl ^- ) manufactured by Wako Pure Chemical Industries, Ltd. is used] and necessary gradient elution [for example, (gradient elution with 0→1.0M NaCl)] to obtain a fraction (FA-1).

By purifying FA-1 by subjecting it to a gel filtration method, that is, a gel filtration method using, for example, Sephadex G-50 manufactured by Wako Pure Chemical Industries, Ltd. and the necessary gradient elution [for example, gradient elution with 0 → 2.0 M NaCl)] to obtain FA-1a, the peptide heteroglycan of the present invention.

(3) A cell wall fragment of Chlorella pyrenoidosa that can be used for producing the peptide heteroglycan of the present invention can be obtained, for example, as follows. That is, first, a chlorella powder/water suspension having a chlorella concentration of 10 % to 25% by weight is adjusted to 10° C. or lower. Next, this suspension is fed into a continuous wet pulverizer as described below and pulverized so that the slurry immediately after pulverization has a temperature of 40° C. or lower. Then, the chlorella slurry thus obtained is immediately cooled to 10° C. or less, whereby chlorella with crushed cell walls can be obtained without deterioration in quality.

The continuous wet pulverizer consists of a large number of glass beads with a diameter of 0.5 mm to 1.5 mm enclosed in a closed cylinder with a cooling jacket. The glass bead volume is 80 % to 85% of the closed cylinder volume, and the glass beads are mixed with the inflow liquid and rotated to grind material in the inflow liquid.

Chlorella whose cell walls have been crushed in this way can be used as it is, but it may also be used after being subjected to an appropriate treatment such as vacuum drying followed by pulverization.

(4) The hematopoietic progenitor cell activator and hematopoietic disorder recovery agent of the present invention can be applied, for example, by oral administration or intraperitoneal administration, but the administration method is not necessarily limited to these. The administration form is not particularly limited, but for example, powders, tablets, hard capsules, soft capsules, liquids dissolved in water, or other liquids can be used.

Various excipients, binders, disintegrants, lubricants, coating agents, coloring agents, corrigents, corrigents, plasticizers and the like can be appropriately used to form various forms.

Examples of excipients include sugars (lactose, white sugar, glucose, mannitol), starch (potato, wheat, corn), inorganic substances (calcium carbonate, calcium sulfate, sodium hydrogen carbonate, sodium chloride), crystalline cellulose, plant powder ( Glycyrrhiza powder, gentian powder) and the like can be mentioned.

Examples of binders include starch paste, gum arabic, gelatin, sodium alginate, methylcellulose (MC), ethylcellulose (EC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hydroxypropylcellulose (HPC), carboxymethylcellulose. (CMC) and the like.

Examples of disintegrants include starch, agar, gelatin powder, crystalline cellulose, CMC·Na, CMC·Ca, calcium carbonate, sodium hydrogen carbonate, sodium alginate and the like.

Examples of lubricants include magnesium stearate, talc, hydrogenated vegetable oils, macrogol, and silicone oils.

Examples of coating agents include sugar coating (white sugar, HPC, shellac), glue coating (gelatin, glycerin, sorbitol), film coating [hydroxypropylmethylcellulose (HPMC), EC, HPC, PVP], enteric coating [hydroxypropylmethylcellulose phthalate (HPMCP), cellulose acetate phthalate (CAP)] and the like.

Examples of coloring agents include water-soluble food dyes and lake dyes. Examples of flavoring agents include lactose, sucrose, glucose, mannitol, and the like. Examples of flavoring agents include aromatic essential oils), light shielding agents (titanium oxide), and the like. Examples of plasticizers include phthalates, vegetable oils, polyethylene glycol) and the like.

The dose of the radiation-induced hematopoietic injury recovery agent of the present invention for humans can be, for example, about 0.83 mg/kg per day.

Tests were conducted on the hematopoietic progenitor cell activating effect and radiation hematopoietic injury recovery effect of the peptide heteroglycan of the present invention.

1. test substance

(1) A closed cylinder with a cooling mantle contains a large number of glass beads with a diameter of 0.5 mm to 1.5 mm having a volume of 80 % to 85% of the volume of the closed cylinder. Chlorella pyrenoidosa concentration 10 Chlorella-pyrenoid sa powder/water suspension of 25% by weight to 25% by weight is fed, pulverized so that the slurry immediately after crushing is 40 ° C. or less, and then the chlorella-pyrenoid obtained in this way. The suspension slurry was immediately cooled to 10° C. or less, vacuum dried, and pulverized to obtain a dry powder of cell wall-crushed Chlorella pyrenoidosa.

(2) The resulting dry powder of crushed cell walls of Chlorella pyrenoidosa was suspended in water and then boiled at 95 °C to 100°C for 2 hours.

After cooling to room temperature, the supernatant was ultrafiltered using a diafilter G-10T (manufactured by Nippon Vacuum Engineering Co., Ltd.: molecular weight cut off: 10,000) while still standing to obtain a first filtration residue.

The precipitate in the stationary state is again boiled with water at 95 ° C. to 100 ° C. for 2 hours, cooled to room temperature, and the supernatant is filtered by a diafilter G-10T (Japan Vacuum Engineering Co., Ltd.) in a stationary state. The second filter residue was obtained by ultrafiltration using a product with a molecular weight cut off of 10,000, and the first filter residue and the second filter residue were mixed.

The mixture was allowed to stand at 3° C. for 12 hours, then centrifuged at room temperature at 10,000 rpm for 20 minutes to separate the supernatant and precipitate.

The supernatant was recovered and ultrafiltered at 3°C using a diafilter G-10T (manufactured by Nippon Vacuum Engineering Co., Ltd.: molecular weight cut off: 10,000) to separate an unfiltered fraction (M.W. 10,000 or more) and a filtered fraction. (M.W. less than 10,000).

Three times the volume of absolute ethanol was added to the non-filtered fraction and centrifuged at 10,000 rpm at room temperature to separate the supernatant and precipitate.

The precipitate was washed three times with absolute ethanol and then once with absolute ether. After that, it was vacuum-dried at 40° C. to obtain FA (crude polysaccharide).

FA was dissolved in water and subjected to ion-exchange chromatography [DEAE-Cellulose (Cl ⁻ ) manufactured by Wako Pure Chemical Industries, Ltd.] and gradient elution [(gradient elution with 0 → 1.0 M NaCl)] to obtain fraction FA. Fractionated into -1, FA-2 and FA-3.

Fraction FA-1 was purified by gel filtration [using Sephadex G-50 manufactured by Wako Pure Chemical Industries, Ltd.] and gradient elution (gradient elution with 0→2.0 M NaCl) to obtain FA-1a. .

The yields of crude polysaccharide FA and peptide heteroglycan FA-1a of the present invention from 400 g of Chlorella pyrenoidosa dry powder were 15.63 g and 253.27 mg, respectively, as vacuum freeze-dried powder.

As a result of analyzing FA-1a as described below, it was found to be a peptide heteroglycan (also simply referred to as "peptide heteroglycan") having the following physicochemical properties (a), (b), (c), (d) and (e) . ).

(i) Analysis of sugar composition

Total sugar was analyzed by the phenol-sulfuric acid method (colorimetric method).

Analysis of constituent sugar composition was carried out as follows.

The specimen FA-1a (2 mg) was dissolved in dimethylsulfoxide (DMSO, 0.5 mL), and sodium hydroxide (20 mg) and methyl iodide (0.2 mL) were added to the solution.

After stirring, the mixture was extracted with chloroform (1.5 mL) and dried under reduced pressure to obtain a methylated polysaccharide.

To this, 80% formic acid (1 mL) was added, dried under reduced pressure, and methylated polysaccharide was obtained with 2N trifluoroacetic acid (1 mL).

After adding 80% formic acid (1 mL) and drying under vacuum, the methylated polysaccharide was hydrolyzed with 2N trifluoroacetic acid (1 mL) and dried under vacuum to obtain a partially methylated polysaccharide.

A small amount of distilled water and sodium borohydride (5 mg) were added to this partially methylated polysaccharide, and the mixture was allowed to stand overnight, after which the sodium borohydride was decomposed with acetic acid.

Further removal of boric acid gave partially methylated alditols. A mixed solution (1 mL) of acetic anhydride/pyridine (1:1) was added thereto, and the mixture was heated for 4 hours and then cooled to dryness. Subsequently, chloroform (2 mL) and distilled water (1 mL) were added and mixed, then the chloroform layer was recovered, anhydrous sodium sulfate (1.5 g) was added, dehydration was performed, and the mixture was dried under reduced pressure.

This was dissolved in 200 μL of distilled water, and 30 μL of the solution was subjected to high performance liquid chromatography (HPLC) analysis (Shimadzu LC-9A) to identify and quantify constituent sugars.

(ii) determination of total nitrogen;

Measured by the semi-micronaldahl method.

(iii) Protein quantification

Protein quantification was performed by the Lowry method.

(iv) amino acid analysis

A test sample (1 mg) was sealed with 6N hydrochloric acid (1 mL), hydrolyzed at 110°C for 20 hours, and subjected to a Hitachi 835 automatic amino acid analyzer.

(v) Measurement of average molecular weight

The average molecular weight was measured by a gel filtration method (Sephadex G-100, Wako Pure Chemical Industries).

As polysaccharide molecular weight markers for gel filtration, α-1,6-linked linear polysaccharide pullulan kit Shodex standard P-82 (Showa Denko) and standard dextran (Sigma-Aldrich) were used.

(vi) Measurement of specific rotation

For the measurement of the specific optical rotation, a circular wood spectroscopic automatic polarimeter (DIP-360 type) was used, and the measurement temperature was 25°C.
(a) average molecular weight: 1 million
(b) Specific rotation: [α] _D -11.6 (Measurement temperature 25°C)
(c) nitrogen content 5.39%, protein content 29.5%, polysaccharide content 70.3%;
(d) Sugar composition (molar ratio): Gal:GIc:Man:Xyl:Rha:Ara:Fru=32:25:13:8:7:5:3
(e) Amino acid composition (mol%):
Glu 15.1
Asp 13.8
Ala 9.9
Leu 8.9
Thr 6.5
Lys 6.2
Va1 5.9
Gly 5.8
Pro 5.5
Ser 5.4
Arg 4.8
Phe 3.6
Ileu 3.1
Tyr 2.1
His 1.7
Met 1.6
Cys 0.1
Total 100%

2. test animal

Six-week-old ICR/Slc male mice obtained from Japan SLC Co., Ltd. were preliminarily bred for seven days under the following breeding conditions. submitted for testing.

3. breeding conditions

Test animals are 10 animals per group, 5 animals each in a plastic cage under a barrier system environment with a temperature of 23 ± 2°C and a relative humidity of 55 ± 5% according to the experimental guidelines of the animal facility of the Mie University Life Science Research Support Center. They were allowed to live together and freely ingest solid feed (CLEA CE-7 manufactured by CLEA Japan, Inc.) and tap water.

4. Radiation irradiation method and irradiation dose

(1) Irradiation method

Using a PHILIPS MG 226/4.5 (a high-precision X-ray generator manufactured by Philips Japan), under the irradiation conditions of a tube voltage of 200 KV, a tube current of 9 mA, and a dose rate of 0.365 Gy/min, each test animal was placed on an acrylic plate. The whole body was irradiated with X-rays while being placed in an irradiation capsule and rotated horizontally.

(2) Examination of irradiation dose

To determine the dose sufficient to induce immunosuppression in the test animals, three groups (10 animals each) were subjected to total body irradiation of 2.5 Gy, 5.0 Gy, and 7.0 Gy, respectively.

7.In the OGy irradiation group, one patient died 11 days after irradiation, and the average survival time was 16.4±3.2. Necropsy of dead mice showed occasional weight loss and atrophy, especially in the thymus and spleen.

5. All 10 cases survived in both the OGy irradiation group and the 2.5Gy irradiation group. Therefore, in this experiment, 5.OGy irradiation was adopted.

5. Irradiation and test substance administration

Using the following 3 groups (10 animals each) of test animals (7-week-old mice above), the activation of hematopoietic progenitor cells by the test substance (peptide heteroglycan) and the recovery effect of radiation-induced hematopoietic damage were examined. did

(a) 5.OGy whole body irradiation/peptide heteroglycan administration group

5. From 24 hours after OGy whole-body irradiation, the dosage of peptide heteroglycan is 10 mg/kg/day, and the distilled aqueous solution of peptide heteroglycan is administered at a rate of 0.2 ml/10 g of body weight twice a day (morning and evening). The rats were intraperitoneally administered every day for 10 days, and raised under the above breeding conditions on the day of irradiation and for 10 days thereafter.

(b) 5.0 Gy total body irradiation group (no peptide heteroglycan administration)

5. From 24 hours after OGy whole body irradiation, physiological saline was intraperitoneally administered twice a day (morning and evening) at a rate of 0.2 ml/10 g of body weight for 10 consecutive days. The animals were bred under the breeding conditions described above.

(c) Non-irradiated group (0 Gy)

At the same timing as in (b) above, physiological saline was intraperitoneally administered at a rate of 0.2 ml/10 g body weight twice a day (morning and evening) for 10 consecutive days. They were bred under the breeding conditions described above for a period of time.

6. Mature blood cell level and number of hematopoietic progenitor cells

(1) Measurement

(1-1) Mature blood cell level

On the next day after the 10-day feeding, heparinized peripheral blood was collected from the posterior aorta of each individual in groups (a), (b) and (c) under ether anesthesia and subjected to measurement.

Red blood cell count, platelet count and white blood cell count were measured with a fully automatic multi-item analyzer (manufactured by JEOL Ltd.).

The granulocyte count, lymphocyte count, and monocyte count were measured under a microscope after preparing a blood smear and Giemsa staining.

(1-2) Number of hematopoietic progenitor cells

After the test animal was euthanized by cervical dislocation under ether anesthesia, the femur, bone marrow and spleen were extracted and used for the preparation of spleen cell suspension and bone marrow cell suspension according to Niho's method.

After the excised spleen was transferred to Eagle's MEM medium previously cooled to 4° C., the spleen was crushed using two glass slides to obtain a splenocyte suspension. Furthermore, a single-cell suspension was obtained by filtration using a stainless steel mesh, and used for culture or detection of antibody-producing cells.

In addition, the bone marrow of one excised femur was suspended in DMEM medium (manufactured by Nissui Pharmaceutical Co.) containing 20% fetal bovine serum (manufactured by U.S.A. St. Louis Mo.Sigma Chemical Co.), Bone marrow (nucleated) cell counts were performed.

[i] Measurement of BFU-E (Erythroid burst_forming cell, early erythroid progenitor cell)

Splenocytes and bone marrow cells in the splenocyte suspension and bone marrow cell suspension were added at a concentration of 2×10 ⁶ cells/ml, respectively, to 2 U/ml using a 35 mm diameter plastic petri dish (USA Falcon™ No 1008). After culturing by the methylcellulose method in the presence of EPO (manufactured by Mo. St. Louis Sigma Chemical Co., USA, recombinant human recombinant erythropoietin), the number of cells was measured.

[ii] CFU-E (Colony-forming unit-erythropoetin dependent, erythropoietin-dependent monocytic progenitor/late erythroblastic progenitor)

Splenocytes and bone marrow cells in the splenocyte suspension and bone marrow cell suspension were added at a concentration of 2×10 ⁵ cells/ml, respectively, using a 35 mm diameter plastic petri dish (USA Falcon (trademark) No 1008) at 2 U/ml. After culturing by the methylcellulose method in the presence of EPO (manufactured by Mo. St. Louis Sigma Chemical Co., USA, recombinant human recombinant erythropoietin), the number of cells was measured.

[iii] CFU-GM (Granulocytes-macrophage colony-forming cell) cell count measurement

The spleen cells and bone marrow cells in the spleen cell suspension and bone marrow cell suspension were added to RPMI 1640 medium containing 1% Pokeweed mitogen ( ^USANY Grand Island GIBCO (trademark)) and 10% fetal bovine serum at a concentration of 2xlO5 cells/ml, respectively. (manufactured by USA St. Louis Mo. Sigma Chemical Co.) and cultured at 37° C. under 5% CO ₂ for one week, and then the number of cells was measured.

(2) Results

Table 1 shows the measurement results.

［表１において、BFU-E（前期赤芽球系前駆細胞）、CFU-E（後期赤芽球系前駆細胞）、CFU-GM（顆粒球マクロファージコロニー形成細胞）。数値は平均±S.E.として示す（n=10）。# P<0.05：非照射群(c)と比較して有意差あり。* P<0.05：5.0Gy全身照射群(b)と比較して有意差あり。］

[In Table 1, BFU-E (early erythroid progenitor cells), CFU-E (late erythroid progenitor cells), and CFU-GM (granulocyte macrophage colony-forming cells). Values are shown as mean±SE (n=10). # P<0.05: Significant difference compared to non-irradiated group (c). * P<0.05: Significant difference compared to 5.0 Gy total body irradiation group (b). ]

(2-1) 5.0 Gy total body irradiation group (b) (peptide heteroglycan non-administration group)

Peripheral red blood cell count, platelet count, peripheral white blood cell count, granulocyte count, lymphocyte count, monocyte count, and BFU-E pro-erythroblastic progenitor cells in bone marrow and spleen compared to non-irradiated group (c) CFU-E late erythroid progenitor cell count, and CFU-GM granulocyte-macrophage colony-forming cell count were all significantly lower.

(2-2) 5. OGy whole body irradiation/peptide heteroglycan administration group (a)

The peripheral red blood cell count, platelet count, peripheral white blood cell count, granulocyte count, lymphocyte count, and monocyte count were all significantly higher than in the 5.0 Gy total body irradiation group (b).

The number of CFU-E late erythroblastic progenitor cells in the bone marrow and spleen was significantly higher than that in the 5.0 Gy total body irradiation group (b) and approached that in the non-irradiation group (c).

Furthermore, the number of BFU-E early erythroid progenitor cells and the number of CFU-GM granulocyte-macrophage colony-forming cells in the bone marrow and spleen were both significantly higher than in the 5.0 Gy whole body irradiation group (b), and the non-irradiation group It was more than (c).

7. histological findings

On the day after the 10 days of breeding, the spleens and thymuses of 3 animals in each group were excised, fixed with 10% formalin at 4°C, and then 4 µm paraffin sections were prepared by a conventional method and stained with hematoxylin and eosin. went and watched. The results are as follows.

(1) Thymus

(1-1) 5.0 Gy whole body irradiation group (b) (Fig. 1)

Lymphocyte destruction was strong, and nuclear debris and nuclear pyknotic cells were scattered, and instead, large reticular cells proliferated. Lymphocytes decreased, the cortex-medullary boundary became unclear, and some images were replaced by reticular cells.

(1-2) 5. OGy whole body irradiation/peptide heteroglycan administration group (a) (Fig. 2)

The thymus weight was significantly larger than that of the 5.0Gy total body irradiation group (b), and histologically, there was a stronger tendency to recover the lymphocyte count than that of the group (b), in which lymphocytes were decreased by irradiation. Admitted.

(2) Spleen

(2-1) 5.0 Gy whole body irradiation group (b) (Fig. 3)

Lymphocytopenia was observed in the white pulp of the spleen, and the lymphoid follicles shrank. The red spleen showed marked hemosiderin pigmentation, which was probably caused by destruction of red blood cells.

(2-2) 5. OGy whole body irradiation/peptide heteroglycan administration group (a) (Fig. 4)

The spleen weight was significantly larger than that of the 5.0 Gy total body irradiation group (b), and histologically, almost no hemosiderin pigmentation due to destruction of red blood cells by irradiation was observed, and vigorous recovery of hematopoietic foci was observed. rice field.

8. As described above, administration of a test substance (peptide heteroglycan) to test animals exposed to irradiation, which causes hematopoietic disorders, revealed that CFU-GM, macrophage hematopoietic progenitor cells, and undifferentiated erythroid hematopoietic progenitor cells BFU-E cells, relatively differentiated erythroid progenitor cells CFU-E, and peripheral erythrocytes showed marked increases. A reduction in disability was noted.

In this specification, the concentration of the aqueous solution is, in principle, mass volume %. Statistical processing was performed by Student's t-test, and a significant difference of less than 5% was determined.

Claims (11)

A hematopoietic progenitor cell activator comprising a peptide heteroglycan having the following physicochemical properties (a), (b), (c), (d) and (e) .
(a) average molecular weight: 1 million
(b) Specific rotation: [α] _D -11.6 (Measurement temperature 25°C)
(c) nitrogen content 5.39%, protein content 29.5%, polysaccharide content 70.3%;
(d) Sugar composition (molar ratio): Gal:GIc:Man:Xyl:Rha:Ara:Fru=32:25:13:8:7:5:3
(e) Amino acid composition (mol%):
Glu 15.1
Asp 13.8
Ala 9.9
Leu 8.9
Thr 6.5
Lys 6.2
Va1 5.9
Gly 5.8
Pro 5.5
Ser 5.4
Arg 4.8
Phe 3.6
Ileu 3.1
Tyr 2.1
His 1.7
Met 1.6
Cys 0.1
Total 100% 2. The hematopoietic progenitor cell activator according to claim 1 , wherein said peptide heteroglycan is derived from Chlorella pyrenoidosa. The hematopoietic progenitor cell activator according to claim 1 or 2 , wherein the hematopoietic progenitor cells are erythropoietic progenitor cells, erythropoietin-dependent monocyte progenitor cells, and neutrophil-granulocyte progenitor cells. A hematopoietic disorder recovery agent comprising a peptide heteroglycan having the following physicochemical properties (a), (b), (c), (d) and (e) .
(a) average molecular weight: 1 million
(b) Specific rotation: [α] _D -11.6 (Measurement temperature 25°C)
(c) nitrogen content 5.39%, protein content 29.5%, polysaccharide content 70.3%;
(d) Sugar composition (molar ratio): Gal:GIc:Man:Xyl:Rha:Ara:Fru=32:25:13:8:7:5:3
(e) Amino acid composition (mol%):
Glu 15.1
Asp 13.8
Ala 9.9
Leu 8.9
Thr 6.5
Lys 6.2
Va1 5.9
Gly 5.8
Pro 5.5
Ser 5.4
Arg 4.8
Phe 3.6
Ileu 3.1
Tyr 2.1
His 1.7
Met 1.6
Cys 0.1
Total 100% 5. The hematopoietic disorder recovery agent according to claim 4 , wherein the peptide heteroglycan is derived from Chlorella pyrenoidosa. Recovery from the hematopoietic disorder includes an increase in the number of erythrocytes, platelets, leukocytes, granulocytes, lymphocytes and monocytes in peripheral blood, an increase in spleen weight, recovery of splenic hematopoietic foci, an increase in thymus weight, and thymic lymphocytes. 6. The hematopoietic disorder recovery agent according to claim 4 or 5 , which is manifested in the recovery of counts. 7. The hematopoietic disorder recovery agent according to any one of claims 4 to 6, wherein the hematopoietic disorder is caused by irradiation. Chlorella pyrenoidosa cell wall crushed product is extracted with hot water,
obtaining a liquid from which low-molecular-weight substances having a molecular weight of 10,000 or less have been removed from the resulting extract;
Absolute ethanol is added to this liquid to obtain a crude polysaccharide as a precipitate,
the resulting crude polysaccharide is dissolved in water and subjected to ion exchange chromatography and gradient elution as required to fractionate;
By purifying one of the obtained fractions, a hematopoietic progenitor cell activator consisting of a peptide heteroglycan having the following physicochemical properties (a), (b), (c), (d) and (e) how to manufacture
(a) average molecular weight: 1 million
(b) Specific rotation: [α] _D -11.6 (Measurement temperature 25°C)
(c) nitrogen content 5.39%, protein content 29.5%, polysaccharide content 70.3%;
(d) Sugar composition (molar ratio): Gal:GIc:Man:Xyl:Rha:Ara:Fru=32:25:13:8:7:5:3
(e) Amino acid composition (mol%):
Glu 15.1
Asp 13.8
Ala 9.9
Leu 8.9
Thr 6.5
Lys 6.2
Va1 5.9
Gly 5.8
Pro 5.5
Ser 5.4
Arg 4.8
Phe 3.6
Ileu 3.1
Tyr 2.1
His 1.7
Met 1.6
Cys 0.1
Total 100% 9. The method of claim 8, wherein the hematopoietic progenitor cells are erythroblastic progenitor cells, erythropoietin-dependent monocytic progenitor cells, and neutrophil-granulocyte progenitor cells. Chlorella pyrenoidosa cell wall crushed product is extracted with hot water,
obtaining a liquid from which low-molecular-weight substances having a molecular weight of 10,000 or less have been removed from the resulting extract;
Absolute ethanol is added to this liquid to obtain a crude polysaccharide as a precipitate,
the resulting crude polysaccharide is dissolved in water and subjected to ion exchange chromatography and gradient elution as required to fractionate;
By purifying one of the obtained fractions, a hematopoietic injury recovery agent consisting of a peptide heteroglycan having the following physicochemical properties (a), (b), (c), (d) and (e) is produced. how to.
(a) Average molecular weight: 1 million
(b) Specific rotation: [α] _D -11.6 (Measurement temperature 25°C)
(c) nitrogen content 5.39%, protein content 29.5%, polysaccharide content 70.3%;
(d) Sugar composition (molar ratio): Gal:GIc:Man:Xyl:Rha:Ara:Fru=32:25:13:8:7:5:3
(e) Amino acid composition (mol%):
Glu 15.1
Asp 13.8
Ala 9.9
Leu 8.9
Thr 6.5
Lys 6.2
Va1 5.9
Gly 5.8
Pro 5.5
Ser 5.4
Arg 4.8
Phe 3.6
Ileu 3.1
Tyr 2.1
His 1.7
Met 1.6
Cys 0.1
Total 100% Recovery from the hematopoietic disorder includes an increase in the number of erythrocytes, platelets, leukocytes, granulocytes, lymphocytes and monocytes in peripheral blood, an increase in spleen weight, recovery of splenic hematopoietic foci, an increase in thymus weight, and thymic lymphocytes. 11. The method of claim 10, which is manifested in recovery of numbers.

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