An immune secretory polypeptide derived from autologous stem cells and method

Abstract

The present invention belongs to the field of biotechnology, and is an immune secretory polypeptide and method derived from autologous stem cells, wherein the polypeptide comprises a polypeptide as shown in SEQ ID NO.1-SEQ ID NO.5. The polypeptide is based on peripheral blood stem cells, etc., and cell secretory bodies are obtained by ultra-nano separation and extraction. Unreported small molecule polypeptide sequences are selected through large model prediction, and relevant immune response, toxicology and anti-tumor tests are carried out. The results show that the sequence will cause specific T cell factors to produce corresponding factor expression responses, and the secreted cytokines have therapeutic effects such as tissue repair and immune regulation.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to an immune secretory polypeptide derived from autologous stem cells and a method thereof. Background Art

[0002] Stem cell therapy is a revolutionary medical method with great prospects in all areas of healthcare. There are different types of stem cell therapy, each with its own unique characteristics and applications. However, due to differences in physical constitution, there may be pain during the injection process, or side effects such as skin allergies. After injection, there is little risk of immune system decline and vascular embolism. In current clinical studies, there is a lot of evidence that mesenchymal stem cells mainly regulate the inflammatory environment in the body through paracrine effects. In addition to the secretion of cytokines, the paracrine effect is also achieved through the secretion of vesicles such as exosomes secreted by mesenchymal stem cells. Exosome therapy is also a modern medical care and regenerative medicine method. The proteins expressed in the extracted exosomes that promote the regeneration of nerve axons are many, and have good tissue compatibility and extremely low immune rejection reactions. They can greatly exert the therapeutic effects of stem cell therapy, while also avoiding the related adverse reactions caused by stem cell transplantation. Therefore, its regenerative ability has great therapeutic potential. Exosomes derived from human autologous gene stem cells are less effective in immune regulation, and there is no clear drug target for screening. Therefore, it is urgent to carry out research in this area. Summary of the invention

[0003] The purpose of the present invention is to prepare an immune secretory polypeptide derived from autologous stem cells, characterized in that the polypeptide comprises polypeptides as shown in SEQ ID NO.1-SEQ ID NO.5.

[0004] The polypeptide preferably has an amino acid sequence prepared by extracting autologous umbilical cord blood as a raw material: SEQ ID 1: LGLLGSDSSASLKWKQGLGPAAIKGLRAASAGPWFSPVG.

[0005] The polypeptide preferably has an amino acid sequence prepared by extracting autologous subcutaneous fat tissue as a raw material: SEQ ID2: GLRALGPISKPIPDPLLGPWKGWFAPLPLLGPTSPGSWLNVVL.

[0006] The polypeptide preferably has an amino acid sequence prepared by extracting autologous bone marrow as a raw material: SEQ ID 3: SPPIIPDWIEDAWFSPGKAVGPAVISKWFWPVEAFP.

[0007] The polypeptide preferably has an amino acid sequence prepared by extracting autologous testicles as raw material: SEQ ID 4: PSKALLEPISKGSPKSPEAPVSKLGEPGLEKLGAD.

[0008] The polypeptide is preferably prepared by extracting autologous peripheral blood as raw material, and the amino acid sequence is: SEQ ID 5: FPPEASRQGKPEAAADEASRQGPKSPAWPKSP.

[0009] The immune secretory polypeptide is preferably derived from autologous stem cells such as umbilical cord blood, subcutaneous fat tissue, bone marrow, testis, and peripheral blood.

[0010] The method comprises the following steps: (1) culturing the raw stem cells in a serum-free medium for 2 to 3 days, collecting the culture supernatant, centrifuging, collecting the supernatant, and obtaining a crude human umbilical cord mesenchymal stem cell exosome fluid;

[0011] (2) The crude stem cell exosome solution in step (1) is concentrated by ultrafiltration and centrifugation to obtain a concentrated solution; the ultrafiltration membrane used for ultrafiltration is 200,000 Da; the centrifugation speed is 2000-2500 g; and the centrifugation time is 25-30 min;

[0012] (3) concentrating the solution in step (2) to obtain a crude product of small molecule active peptides from human umbilical cord mesenchymal stem cell exosomes;

[0013] (4) Dissolve the crude stem cell exosome small molecule active peptide in (3) in water to prepare a solution with a concentration of 200 mg / mL. The invention relates to separation and purification by LH-20 column chromatography, wherein the mobile phase is 20% methanol, the flow rate is 0.1-0.3 mL / min, the absorbance of the eluent is measured at 250 nm or 200 nm, and the desired peak is collected according to the absorbance value; the invention relates to further purification by high performance liquid chromatography, and the chromatographic conditions are as follows: C18 chromatographic column, the mobile phase A is water containing 0.1-0.15% trifluoroacetic acid by volume, the mobile phase B is acetonitrile, the gradient elution conditions are: 0-25 min, 3% B, 25-30 min, 5%-10% B, 25-30 min, 10% B-20% B, 30-40 min, 20% B-35% B, the flow rate is 2.0 mL / min, the detection wavelength is 250 or 280 nm, the chromatographic peak with a retention time of 40 minutes is collected, and the small molecule active peptide is obtained by freeze drying after concentration; the small molecule peptide collected for purity and amino acid sequence determination is a single peak after liquid chromatography detection, and the structural sequence is determined by high performance liquid chromatography-mass spectrometry.

[0014] Furthermore, the present application also protects the use of an immunologically secreted polypeptide in the preparation of immunomodulatory drugs.

[0015] Furthermore, the medicine preferably includes capsules, aqueous emulsions, compressed tablets, nasal inhalation types, etc.

[0016] The technical effects achieved by the present invention are:

[0017] (1) The present invention establishes a method system that can use autologous peripheral blood stem cells to secrete exosome small molecule polypeptides, and the method is simple and easy to operate;

[0018] (2) The various peptides isolated and identified are safe, easily absorbed, and non-toxic to macrophages within a concentration range of 5-200 μg / mL.

[0019] (3) The isolated and identified small molecule peptides can significantly release immune factors such as CD31, TNF-α, IL-6, NO, and CD45 within a concentration range of 5-200 μg / mL; the tumor inhibition rate increases to 98% with the increase of the small molecule peptide dose, indicating that the small molecule active peptide has a significant effect in inhibiting the growth of lung cancer tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : Toxicological evaluation test chart;

[0021] Figure 2 : Immune factor overexpression test chart;

[0022] Figure 3 : Tumor inhibition graph of treatment group;

[0023] Figure 4 : Protein sequence blast analysis results. DETAILED DESCRIPTION

[0024] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the accompanying drawings.

[0025] Embodiment 1

[0026] The source of the immune secretory polypeptide is preferably stem cells such as peripheral blood.

[0027] (1) culturing the raw stem cells in a serum-free medium for 2 hours, collecting the culture supernatant, centrifuging, and collecting the supernatant to obtain a crude stem cell exosome solution;

[0028] (2) The crude human umbilical cord mesenchymal stem cell exosomes in step (1) is concentrated by ultrafiltration and centrifugation to obtain a concentrated solution; the ultrafiltration membrane used for ultrafiltration is 200000Da; the centrifugation speed is 2000g; and the centrifugation time is 25min;

[0029] (3) concentrating the solution in step (2) to obtain a crude product of small molecule active peptides from human umbilical cord mesenchymal stem cell exosomes;

[0030] (4) Dissolving the crude product of stem cell exosome small molecule active peptide in (3) in water to prepare a solution with a concentration of 200 mg / mL, separating and purifying it by Sephadex LH-20 column chromatography, the mobile phase is 20% methanol, the flow rate is 0.1 mL / min, the absorbance of the eluent is measured at 250 nm, and the desired peak is collected according to the absorbance value; further purifying by high performance liquid chromatography, the chromatographic conditions are as follows: C18 column, mobile phase A is water containing 0.1% trifluoroacetic acid by volume, mobile phase B is acetonitrile, gradient elution conditions are: 10 min, 3% B, 25 min, 5% B, 25 min, 10% B, 30 min, 20% B, the flow rate is 2.0 mL / min, the detection wavelength is 250 nm, the chromatographic peak with a retention time of 40 minutes is collected, and the small molecule active peptide is obtained after concentration and freeze-drying;

[0031] Purity and amino acid sequence determination The collected small molecule peptides were detected as a single peak by liquid chromatography, and the structural sequence was determined by high performance liquid chromatography-mass spectrometry.

[0032] Embodiment 2

[0033] The sources of immune secretory polypeptides are preferably subcutaneous fat tissue, umbilical cord blood, bone marrow, and testicular raw stem cells.

[0034] Step (1) culturing the raw stem cells in a serum-free medium for 3 days, collecting the culture supernatant, centrifuging, and collecting the supernatant to obtain a crude stem cell exosome solution;

[0035] (2) The crude human umbilical cord mesenchymal stem cell exosomes in step (1) is concentrated by ultrafiltration and centrifugation to obtain a concentrated solution; the ultrafiltration membrane used for ultrafiltration is 200000Da; the centrifugation speed is 2500g; and the centrifugation time is 30min;

[0036] (3) concentrating the solution in step (2) to obtain a crude product of small molecule active peptides from human umbilical cord mesenchymal stem cell exosomes;

[0037] (4) Dissolving the crude product of stem cell exosome small molecule active peptide in (3) in water to prepare a solution with a concentration of 200 mg / mL, separating and purifying it by Sephadex LH-20 column chromatography, the mobile phase is 20% methanol, the flow rate is 0.3 mL / min, the absorbance of the eluent is measured at 200 nm, and the desired peak is collected according to the absorbance value; further purification is performed by high performance liquid chromatography, and the chromatographic conditions are as follows: C18 column, mobile phase A is water containing 0.15% trifluoroacetic acid by volume, mobile phase B is acetonitrile, gradient elution conditions are: 25 min, 3% B, 30 min, 10% B, 30 min, 20% B, 40 min, 35% B, the flow rate is 2.0 mL / min, the detection wavelength is 280 nm, the chromatographic peak with a retention time of 40 minutes is collected, and the small molecule active peptide is obtained after concentration and freeze-drying;

[0038] Purity and amino acid sequence determination The collected small molecule peptides were detected as a single peak by liquid chromatography, and the structural sequence was determined by high performance liquid chromatography-mass spectrometry.

[0039] Embodiment 3

[0040] The small molecule active peptides obtained by concentration and freeze drying in Examples 1 and 2 were further analyzed by UPLC-LTQ-Orbitrap-Velos Pro mass spectrometer, and analyzed and identified by using the mass spectrometry software Compound Discovery. Figure 4 , analysis and identification, identified a variety of peptides, and selected short peptides that are safe and easily absorbed as follows:

[0041] The polypeptide preferably has an amino acid sequence prepared by extracting autologous umbilical cord blood as a raw material: SEQ ID 1: LGLLGSDSSASLKWKQGLGPAAIKGLRAASAGPWFSPVG.

[0042] The polypeptide preferably has an amino acid sequence prepared by extracting autologous subcutaneous fat tissue as a raw material: SEQ ID2: GLRALGPISKPIPDPLLGPWKGWFAPLPLLGPTSPGSWLNVVL.

[0043] The polypeptide preferably has an amino acid sequence prepared by extracting autologous bone marrow as a raw material: SEQ ID 3: SPPIIPDWIEDAWFSPGKAVGPAVISKWFWPVEAFP.

[0044] The polypeptide preferably has an amino acid sequence prepared by extracting autologous testicles as raw material: SEQ ID 4: PSKALLEPISKGSPKSPEAPVSKLGEPGLEKLGAD.

[0045] The polypeptide is preferably prepared by extracting autologous peripheral blood as raw material, and the amino acid sequence is: SEQ ID 5: FPPEASRQGKPEAAADEASRQGPKSPAWPKSP.

[0046] Embodiment 4

[0047] Toxicity evaluation of small molecule peptide pairs in Example 3

[0048] Macrophage density = 1×10 6 / mL, 300 μL per well was inoculated in a 96-well plate, cultured in a 38°C, 5% CO2 incubator for 2D, and different concentrations of the small molecule polypeptide SEQ ID NO.1-SEQ ID NO.5 described in Example 3 were added. (5, 10, 20, 100, 200 μg / mL), the blank control group and the positive control group were added with culture medium and LPS (100 ng / mL) respectively, and cultured for 24 hours; the 96-well plate was taken out, the old culture medium was discarded, and cultured in a 37°C, 5% CO2 incubator for 90 minutes, and then the absorbance at 450nm was measured with an enzyme marker. The results are shown in Table 1. Within the concentration range of 5-200 μg / mL, there was no toxicity to macrophages.

[0049] Table 1 Toxicological evaluation

[0050]

[0051] Effects of small molecule peptides on the expression of genes related to immune factor assessment:

[0052] Macrophage density = 1×10 6 / mL, 300μL per well was inoculated in a 96-well plate, and cultured in a 38°C, 5% CO2 incubator for 2D, and different concentrations of the small molecule polypeptide SEQ ID NO.1-SEQ ID NO.5 of Example 3 were added. (5, 10, 50, 10, 200μg / mL), the control group and the positive control group were added with culture medium and LPS (100ng / mL), and the cell supernatant was collected after 24h of culture for the determination of the production of related immune cytokines. NO was determined using Griess reagent. 100μL of cell supernatant was mixed with an equal volume of Griess reagent, and the absorbance at 542nm was measured with an enzyme marker after standing in the dark for 10 minutes. The cytokine production and gene expression levels were determined by ELASA kit and qRT-PCR technology, respectively. The results are shown in Table 2. Within the concentration range of 5-200μg / mL, CD31, TNF-α, IL-6, NO,

[0053] CD45 factor.

[0054] Table 2 Immune cytokine response test

[0055]

[0056] Determination of anti-tumor activity of small molecule peptides:

[0057] Take the tumor tissue of lung cancer mice with good growth, add physiological saline, and prepare a tumor tissue cell suspension at a ratio of 1:5. Inoculate 0.2 mL subcutaneously in the axilla of the left forelimb of the mouse.

[0058] 24 hours after inoculation, the lung cancer mice were randomly divided into 5 groups (model group and experimental groups 1, 2, 3, 4, 5), with 10 mice in each group. The mice in experimental groups 1, 2, 3, 4, and 5 were gavaged with 100 mg / (kg·d), 200 mg / (kg·d), 300 mg / (kg·d), 400 mg / (kg·d), and 500 mg / (kg·d) of small molecule active peptides, respectively, and the model group was gavaged with an equal amount of normal saline for 120 consecutive days. After 120 days, the mice were weighed with an electronic analytical balance, and then the mice were killed by dislocation, the complete tumor tissue was removed, and the tumor tissue mass of the mice was weighed with an electronic analytical balance, and finally the tumor inhibition rate was calculated.

[0059] The calculation formula of tumor inhibition rate is: tumor inhibition rate (%) = (average tumor weight of model group - average tumor weight of experimental group) / average tumor weight of model group × 100%;

[0060] The experimental results are shown in Table 3. The tumor inhibition rate increases with the increase of the small molecule peptide dose. Among them, the tumor inhibition rate of the 500 mg / (kg·d) dose group is the highest, reaching 98%, indicating that the small molecule active peptide has a significant effect in inhibiting the growth of lung cancer tumors.

[0061] Table 3 Tumor inhibition rate in treatment groups

[0062]

[0063] The above-mentioned small molecule polypeptides have the characteristics of no electrolysis and no ionization. At 80 degrees Celsius and ultra-low temperature environments, their structural performance is stable and their activity remains unchanged. They are pure natural ingredients, without preservatives, hormones, alcohol, or irritating odors. The water chromatography test of this product is blue, and there are no side effects and it can be used safely.

[0064] The above is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be considered as the protection scope of the present invention. The structures, devices and operating methods not specifically described and explained in the present invention shall be implemented according to the conventional means in the art unless otherwise specified and limited.

Claims

1. An immune secretory polypeptide derived from autologous stem cells, characterized in that: The polypeptide is a polypeptide as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, or SEQ ID NO.

5.

2. The immune secretory polypeptide according to claim 1, characterized in that: The polypeptide is an amino acid sequence: SEQ ID 1: LGLLGSDSSASLKWKQGLGPAAIKGLRAASAGPWFSPVG.

3. The immune secretory polypeptide according to claim 1, characterized in that: The polypeptide is an amino acid sequence: SEQ ID 2: GLRALGPISKPIPDPLLGPWKGWFAPLPLLGPTSPGSWLNVVL.

4. The immune secretory polypeptide according to claim 1, characterized in that: The polypeptide is an amino acid sequence: SEQ ID 3: SPPIIPDWIEDAWFSPGKAVGPAVISKWFWPVEAFP.

5. The immune secretory polypeptide according to claim 1, characterized in that: The polypeptide is an amino acid sequence: SEQ ID 4: PSKALLEPISKGSPKSPEAPVSKLGEPGLEKLGAD.

6. The immune secretory polypeptide according to claim 1, characterized in that: The polypeptide is an amino acid sequence: SEQ ID 5: FPPEASRQGKPEAAAADEASRQGPKSPAWPKSP.

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