Medicinal umbilical cord mesenchymal stem cell factor and preparation method thereof

Abstract

The application provides a medicine umbilical cord mesenchymal stem cell factor and a preparation method thereof, belongs to the biotechnology field, and the preparation method comprises the following steps: inducing and culturing the third generation of expanded cells of umbilical cord mesenchymal stem cells, the induction culture medium contains epidermal growth factor, vascular endothelial growth factor, deferoxamine, vitamin C, rapamycin, asiaticoside, astragaloside IV, astragalus polysaccharide and the like, the secretion of the target factor is effectively promoted, cell differentiation is controlled, two kinds of chromatography columns are used for purification and extraction, the product yield is high, the purity is high, and the efficacy of the product is further improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology, and particularly relates to a medicine umbilical cord mesenchymal stem cell factor and a preparation method thereof. BACKGROUND

[0002] Stem cells are a class of cells with self-renewal and multi-directional differentiation potential, which play a key role in tissue repair, immune regulation, etc. Mesenchymal stem cells (MSCs) as an important member, are widely sourced, including bone marrow, fat, umbilical cord, etc. Umbilical cord mesenchymal stem cells (UC-MSCs) have become a research hotspot due to their unique advantages. Compared with bone marrow and other sources, umbilical cord is easy to obtain, does not harm the donor, and has low immunogenicity, greatly reducing the risk of immune rejection, which provides a broad prospect for its clinical application.

[0003] The key to the function of umbilical cord mesenchymal stem cells lies in the various bioactive factors they secrete, i.e. umbilical cord mesenchymal stem cell factors. These factors are complex and include growth factors, cytokines and chemokines. Growth factors such as epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) are crucial in cell proliferation, migration and angiogenesis. EGF can promote the proliferation and differentiation of epidermal cells and accelerate skin wound healing; VEGF is of great significance to angiogenesis and can improve the blood supply of ischemic tissues, and has great potential in the treatment of ischemic diseases such as myocardial infarction and ischemic stroke.

[0004] Cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) play a key role in the immune regulation network. IL-6 is involved in the initiation and regulation of inflammatory response, and appropriate amount of IL-6 can activate immune cells and enhance the body's immune defense; TNF-α plays a role in tumor cell apoptosis induction and immune cell activation. However, abnormal expression of cytokines is also associated with various diseases. In inflammatory diseases, excessive secretion of cytokines can lead to uncontrolled inflammation and cause tissue damage. Cytokines in umbilical cord mesenchymal stem cell factors finely regulate the function of immune cells through complex signal transduction mechanisms and maintain immune balance.

[0005] Chemokines can guide the directional migration of immune cells to the injury or inflammation site, promote the interaction between immune cells and target cells, and enhance the effect of immune response. This precise cell recruitment mechanism enables immune cells to quickly and accurately reach the lesion area and play a therapeutic role in disease treatment.

[0006] However, the current preparation methods of umbilical cord mesenchymal stem cell factors mainly have the following problems:

[0007] (1) The secretion of stem cell factors is difficult to control: precise control of temperature, humidity, oxygen content and other environmental factors is required, and specific culture medium and additives are also required to maintain the normal physiological function of cells. Any slight change will affect cell growth and the secretion of stem cell factors, affecting the yield of target products.

[0008] (2) Difficult to control differentiation: umbilical cord mesenchymal stem cells will differentiate during culture, resulting in loss of stem cell characteristics, which will affect the type and amount of stem cell factor secretion and reduce its application value. SUMMARY

[0009] In view of the above technical problems existing in the preparation of umbilical cord mesenchymal stem cell factors, the present application provides a pharmaceutical umbilical cord mesenchymal stem cell factor and a preparation method thereof. EGF and VEGF are secreted and extracted by using a special induction medium and a purification method, which effectively improves the control of differentiation, improves the secretion and extraction rate of target product factors, and further improves the efficacy of the product. The specific technical scheme is as follows:

[0010] A pharmaceutical umbilical cord mesenchymal stem cell factor, comprising epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF).

[0011] The yield of the umbilical cord mesenchymal stem cell factor is 0.58% or more.

[0012] The preparation method of the above-mentioned pharmaceutical umbilical cord mesenchymal stem cell factor comprises the following steps:

[0013] S1: Take the 3rd generation of expanded cells of umbilical cord mesenchymal stem cells, inoculate into an induction medium, and the cell density is 1×10 6 cells / mL~1×10 7 cells / mL, and perform induction culture;

[0014] The induction medium is an endothelial cell culture medium (EGM-2) containing 20wt%~25wt% FBS (fetal bovine serum), 50ng / mL~60ng / mL epidermal growth factor (EGF), 20ng / mL~30ng / mL vascular endothelial growth factor (VEGF), 50μg / mL~60μg / mL deferoxamine (HIF-1α stabilizer), 50μg / mL~60μg / mL vitamin C (synergistic enhancement of VEGF secretion), 1wt%~2wt% penicillin-streptomycin, 10ng / mL~15ng / mL rapamycin (promotes autophagy through mTOR pathway and maintains cell viability), 30μg / mL~50μg / mL asiaticoside, 30μg / mL~50μg / mL astragaloside, 30μg / mL~50μg / mL astragalus polysaccharide, and the balance is deionized water.

[0015] S2: After the induction culture is completed, the cell culture supernatant is collected; the supernatant is centrifuged to obtain a centrifugal supernatant; the centrifugal supernatant is loaded onto a HiTrap Heparin HP column to capture VEGF; then PBS is used for washing to remove unbound components to obtain a washing liquid A; then an elution buffer containing 0.1M glycine-HCl pH2.5-3.0 is used for elution, and immediately a 1M Tris-HCl pH9.0-9.2 neutralizing solution is used to adjust the pH value of the eluate to 7.0-7.5 to obtain a purified liquid B;

[0016] The washing liquid A is loaded onto a Q Sepharose XL column (pH8.0) to capture EGF; then PBS is used for washing to remove unbound impurities; then an elution buffer containing 0.1M glycine-HCl pH2.5-3.0 is used for elution, and immediately a 1M Tris-HCl pH9.0-9.2 neutralizing solution is used to adjust the pH value of the eluate to 7.0-7.5 to obtain a purified liquid C; the purified liquid B and the purified liquid C are combined to obtain a stem cell factor solution;

[0017] S3: The stem cell factor solution is loaded into a dialysis bag, which is placed in a dialysis solution for dialysis; trehalose is added to the stem cell factor solution after dialysis as a protective agent, and after freeze-drying, the stem cell factor solution is stored at-80℃ to obtain an umbilical cord mesenchymal stem cell factor.

[0018] In S1 of the above preparation method, the induction culture is performed in an incubator at 36.5℃-37.5℃, 5%-6% CO2, and saturated humidity for 7d-10d.

[0019] In S2 of the above preparation method, the supernatant is centrifuged at 3000rpm-3500rpm for 15min-20min to obtain a centrifugal supernatant; the HiTrap Heparin HP column is equilibrated with PBS before loading; the Q Sepharose XL column is equilibrated with PBS before loading; the PBS washing is 3-5 column volumes; and the elution is 2-3 column volumes.

[0020] In S3 of the above preparation method, the molecular weight cut-off of the dialysis bag is 3.5kDa; the dialysis solution contains 10wt%-20wt% PBS; the dialysis is performed at 4℃-5℃ for 12h-24h, during which the dialysis solution is replaced 3-4 times to remove small molecular salts and impurities; and the amount of trehalose added is 4.5wt%-5.5wt% of the mass of the stem cell factor solution.

[0021] In S1 of the above preparation method, the method for obtaining the third generation of expanded cells of the umbilical cord mesenchymal stem cells comprises:

[0022] M1: Put the umbilical cord tissue block into a sterile culture dish, add a sterile aqueous solution containing collagenase II, collagenase IV and trypsin, shake and digest for 50-70 min, add low-sugar DMEM medium containing 10-12 wt% FBS to terminate the digestion, and obtain a cell suspension; filter the cell suspension through a cell screen, centrifuge the filtrate, and collect the cell precipitate;

[0023] M2: Resuspend the cell precipitate with low-sugar DMEM medium containing 10-12 wt% platelet lysate, 1-1.5 wt% Gluta MAX, and 10-12 ng / mL basic fibroblast growth factor (bFGF), adjust the cell density to 1x10 6 7 6 / mL-1x106 / mL, and perform primary culture; when the cell confluence reaches 80-90%, perform subculture;

[0024] M3: Discard the culture medium, rinse the cells with PBS, add 0.25-0.3 wt% trypsin-EDTA digestion solution, incubate at 36.5-37.5°C for 1-2 min, when the cells become round and start to detach, add low-sugar DMEM medium containing 10-12 wt% FBS to terminate the digestion; blow the cells to form a single cell suspension, inoculate in a new culture bottle for continuous culture, and obtain subculture expanded cells;

[0025] M4: Repeat the M3 step until the third generation of expanded cells is obtained.

[0026] In the above method for obtaining the third generation of expanded cells of umbilical cord mesenchymal stem cells, the sterile aqueous solution contains 0.1-0.15 wt% collagenase II, 0.1-0.15 wt% collagenase IV, and 0.05-0.08 wt% trypsin; the temperature for shaking and digestion is 36.5-37.5°C; the cell screen is 70-80 μm; the centrifugation speed of the filtrate is 1000-1200 rpm, and the centrifugation time is 5-8 min.

[0027] In the above method for obtaining the third generation of expanded cells of umbilical cord mesenchymal stem cells, the primary culture is performed in a culture incubator at 36.5-37.5°C, 5-6% CO2, and saturated humidity, and the culture medium is replaced every 3 days until the cell confluence reaches 80-90%.

[0028] In the above method for obtaining the third generation of expanded cells of umbilical cord mesenchymal stem cells, the cells are rinsed with PBS 2-3 times; the volume ratio of inoculation is single cell suspension: medium = 1:(3-4), and the medium is the same as that used for primary culture.

[0029] The umbilical cord mesenchymal stem cell factor for medicine and the preparation method thereof have the following beneficial effects:

[0030] I. In the preparation method, in the cell acquisition stage, collagenase II, collagenase IV and trypsin can effectively separate the cells in the umbilical cord tissue block, and will not cause excessive damage to the cells. In the subsequent cell culture process, the platelet lysate provides rich growth factors and nutrients, GlutaMAX provides a stable source of glutamine for cells, glutamine participates in key physiological processes such as energy metabolism and nucleic acid synthesis of cells, and basic fibroblast growth factor (bFGF) can activate a series of intracellular signaling pathways, promote cell cycle progression, and maintain cell proliferation and stemness. In the induction culture stage, various factors in the induction medium synergistically act, FBS provides rich nutrients, epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) directly participate in the growth and differentiation regulation of cells, deferoxamine acts as a HIF-1 alpha stabilizer, simulates a hypoxic environment, activates intracellular genes related to hypoxic adaptation, promotes the secretion of VEGF and other factors, vitamin C synergistically enhances the secretion of VEGF, penicillin-streptomycin prevents cell contamination, rapamycin promotes autophagy through the mTOR pathway, maintains cell viability, and asiaticoside, astragaloside, and astragalus polysaccharide regulate the cell microenvironment and intracellular signaling pathways, and together promote the secretion of EGF and VEGF. These factors work together to obtain a good yield and high factor content, and significantly promote the proliferation of HUVEC cells.

[0031] II. The basic fibroblast growth factor (bFGF) is an important growth factor, which can bind to the receptor on the cell surface, activate a series of intracellular signaling pathways, Ras-Raf-MEK-ERK signaling pathway, promote cell cycle progression, and make the cells from G1 phase to S phase, thereby promoting cell proliferation. At the same time, bFGF also plays an important role in maintaining stem cell stemness, which can inhibit the differentiation of stem cells and maintain the pluripotency of stem cells.

[0032] III. EGF and VEGF are key regulators of cell growth and differentiation, which can directly act on cell surface receptors, activate intracellular signaling pathways, and promote cell proliferation, migration and differentiation.

[0033] IV. Deferoxamine acts as a HIF-1 alpha stabilizer and can simulate a hypoxic state in the cell microenvironment. Under normal oxygen conditions, HIF-1 alpha is rapidly degraded, but deferoxamine can inhibit its degradation, so that HIF-1 alpha stably exists. Stable HIF-1 alpha can activate a series of intracellular genes related to hypoxic adaptation, including the expression of VEGF and other angiogenesis-related factors.

[0034] V. Rapamycin promotes autophagy through mTOR pathway, autophagy is a self-degradation process in cells, which can remove damaged organelles and protein aggregates, maintain the stability of intracellular environment, ensure the cells in a healthy metabolic state during induction culture, and is conducive to the sustained secretion of stem cell factors. Vitamin C not only participates in the intracellular antioxidant process, but also synergistically enhances VEGF secretion. It can achieve this effect by regulating the intracellular redox state and affecting the activity of VEGF synthesis related enzymes. Rapamycin and vitamin C have good synergistic effect when used together.

[0035] VI. Aspidosperma contains a variety of bioactive ingredients, which can reduce the damage of inflammatory reaction to cells by regulating the immune microenvironment of cells; and can promote cell growth and factor secretion by promoting intercellular communication and enhancing the interaction between cells. Astragalus glycoside and astragalus polysaccharide can enhance the immune function of cells by regulating cell immunity, so that the cells can better cope with various stimuli in the culture environment; and can synergistically promote cell growth and factor secretion by promoting intercellular communication and regulating intracellular signal transduction pathways. The three of them have good synergistic effect on the secretion of EGF and VEGF when used together.

[0036] VII. The step-by-step chromatography of HiTrap Heparin HP column and Q Sepharose XL column can effectively extract and purify the target product, and improve the yield and purity. DETAILED DESCRIPTION

[0037] The application will be further described below in combination with specific implementation examples, but the application is not limited to these examples.

[0038] Example 1

[0039] A drug umbilical cord mesenchymal stem cell factor includes epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF).

[0040] The preparation method of the above-mentioned drug umbilical cord mesenchymal stem cell factor includes the following steps:

[0041] S0, obtaining the third generation of expanded cells of umbilical cord mesenchymal stem cells:

[0042] M1: Put the umbilical cord tissue block into a sterile culture dish, add a sterile aqueous solution containing 0.12wt% collagenase II, 0.12wt% collagenase IV and 0.06wt% trypsin, and shake and digest at 37℃ for 60min, then add low-sugar DMEM medium containing 11wt% FBS to terminate digestion, and obtain a cell suspension; filter the cell suspension through a 70μm cell screen, centrifuge the filtrate at 1100rpm for 6min, and collect the cell precipitate;

[0043] M2: Resuspend the cell pellet with low glucose DMEM medium containing 11 wt% platelet lysate, 1.2 wt% GlutaMAX, 11 ng / mL basic fibroblast growth factor (bFGF), and adjust the cell density to 1 x 10 6 6 cells / mL, and place in an incubator at 37°C, 5% CO2, and saturated humidity. Replace the medium every 3 days, and subculture when the cell confluence reaches 85%.

[0044] M3: Discard the medium, rinse the cells twice with PBS, add 0.28 wt% trypsin-EDTA digestion solution, and incubate at 37°C for 90 s. When the cells become rounded and start to detach, add low glucose DMEM medium containing 11 wt% FBS to terminate the digestion. Blow the cells to form a single-cell suspension, and inoculate the new culture flask at a volume ratio of 1:3.5. Continue to culture in the same medium as used in the primary culture, and obtain the subculture expanded cells.

[0045] M4: Repeat the M3 step until the third-generation expanded cells are obtained.

[0046] S1, induction culture: Take the third-generation expanded cells of the umbilical cord mesenchymal stem cells, and inoculate into the induction medium at a cell density of 1 x 10 6

[0047] The induction medium is endothelial cell medium (EGM-2) containing 22 wt% FBS, 55 ng / mL epidermal growth factor (EGF), 25 ng / mL vascular endothelial growth factor (VEGF), 55 μg / mL deferoxamine (HIF-1α stabilizer), 55 μg / mL vitamin C (synergistically enhances VEGF secretion), 1.5 wt% penicillin-streptomycin, 12 ng / mL rapamycin (promotes autophagy through the mTOR pathway and maintains cell viability), 40 μg / mL asiaticoside, 40 μg / mL astragaloside, 40 μg / mL astragalus polysaccharide, and the balance is deionized water.

[0048] S2, purification and extraction: After the induction culture is completed, collect the cell culture supernatant; centrifuge the supernatant at 3200 rpm for 18 min to obtain the centrifuged supernatant; load the centrifuged supernatant onto a HiTrap Heparin HP column previously equilibrated with PBS to capture VEGF; then rinse the column with PBS for 4 column volumes to remove unbound components, and obtain the rinse liquid A; then elute with 0.1 M glycine-HCl pH 2.8 elution buffer for 2 column volumes, immediately adjust the pH of the eluate to 7.2 with 1 M Tris-HCl pH 9.1 neutralization solution, and obtain the purified liquid B.

[0049] The rinse solution A is loaded onto a Q Sepharose XL column (pH 8.0) previously equilibrated with PBS to capture EGF; then the chromatography column is rinsed with PBS for 4 column volumes to remove unbound impurities; then eluted with elution buffer containing 0.1M glycine-HCl pH 2.7 for 2 column volumes, immediately adjust the pH of the eluate to 7.3 with 1M Tris-HCl pH 9.1 neutralization solution to obtain the purified solution C; combine the purified solution B and the purified solution C to obtain the stem cell factor solution.

[0050] S3, post-treatment: the stem cell factor solution is loaded into a dialysis bag (3.5kDa molecular weight cut-off), placed in a dialysis solution containing 15wt% PBS, dialyzed at 4°C for 18h, during which the dialysis solution is replaced 3 times to remove small molecule salts and impurities; 5wt% trehalose is added to the dialyzed stem cell factor solution as a protective agent, freeze-dried and stored at -80°C to obtain the umbilical cord mesenchymal stem cell factor.

[0051] Example 2

[0052] A pharmaceutical umbilical cord mesenchymal stem cell factor comprising epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF).

[0053] The preparation method of the above-mentioned pharmaceutical umbilical cord mesenchymal stem cell factor comprises the following steps:

[0054] S0, obtaining the 3rd generation expanded cells of umbilical cord mesenchymal stem cells:

[0055] M1: place the umbilical cord tissue block into a sterile culture dish, add a sterile aqueous solution containing 0.1wt% collagenase II, 0.1wt% collagenase IV and 0.05wt% trypsin, shake and digest at 36.5°C for 50min, add low-glucose DMEM medium containing 10wt% FBS to terminate digestion, and obtain a cell suspension; filter the cell suspension through a 70μm cell screen, centrifuge the filtrate at 1000rpm for 5min, and collect the cell precipitate;

[0056] M2: resuspend the cell precipitate with low-glucose DMEM medium containing 10wt% platelet lysate, 1wt% GlutaMAX, 10ng / mL basic fibroblast growth factor (bFGF), adjust the cell density to 1×10 6 6 / mL, and place it in an incubator at 36.5°C, 5% CO2 and saturated humidity; replace the medium every 3 days, and subculture when the cell confluence reaches 80%;

[0057] M3: Discard the culture medium, rinse the cells twice with PBS, add 0.25wt% trypsin-EDTA digestion solution, incubate at 36.5°C for 1 min, when the cells become round and start to detach, add low-glucose DMEM medium containing 10wt% FBS to terminate the digestion; blow the cells to form a single cell suspension, inoculate in a new culture bottle at a volume ratio of 1:3, continue to culture, the culture medium is the same as that used in the primary culture, and the passage expansion cells are obtained;

[0058] M4: Repeat the M3 step until the third generation of expanded cells is obtained.

[0059] S1, induction culture: take the third generation of expanded cells of the umbilical cord mesenchymal stem cells, inoculate in the induction medium, the cell density is 1x10 6 cells / mL; induction culture in a 36.5°C, 5% CO2, saturated humidity incubator for 7 days;

[0060] The induction medium is endothelial cell medium (EGM-2) containing 20wt% FBS, 50ng / mL epidermal growth factor (EGF), 20ng / mL vascular endothelial growth factor (VEGF), 50μg / mL deferoxamine (HIF-1α stabilizer), 50μg / mL vitamin C (synergistic enhancement of VEGF secretion), 1wt% penicillin-streptomycin, 10ng / mL rapamycin (promotes autophagy through the mTOR pathway and maintains cell viability), 30μg / mL asiaticoside, 30μg / mL astragaloside, 30μg / mL astragalus polysaccharide, and the balance is deionized water.

[0061] S2, purification and extraction: after the induction culture is completed, the cell culture supernatant is collected; the supernatant is centrifuged at 3000rpm for 15min to obtain the centrifuged supernatant; the centrifuged supernatant is loaded onto a pre-equilibrated HiTrap Heparin HP column with PBS, and VEGF is captured; then the column is washed with PBS for 3 column volumes to remove unbound components, and the washing liquid A is obtained; then eluted with 0.1M glycine-HCl pH2.5 elution buffer for 2 column volumes, immediately adjust the pH value of the eluate to 7.0 with 1M Tris-HCl pH9.0 neutralizing solution, and the purified liquid B is obtained;

[0062] The washing liquid A is loaded onto a pre-equilibrated Q Sepharose XL column (pH8.0) with PBS, and EGF is captured; then the column is washed with PBS for 3 column volumes to remove unbound impurities; then eluted with 0.1M glycine-HCl pH2.5 elution buffer for 2 column volumes, immediately adjust the pH value of the eluate to 7.0 with 1M Tris-HCl pH9.0 neutralizing solution, and the purified liquid C is obtained; combine the purified liquid B and the purified liquid C to obtain the stem cell factor solution.

[0063] S3, post-processing: the stem cell factor solution is loaded into a dialysis bag (molecular weight cut-off 3.5 kDa), placed in a dialysis solution containing 10 wt% PBS, dialyzed at 4°C for 12 h, during which the dialysis solution is replaced 3 times to remove small molecule salts and impurities; 4.5 wt% trehalose is added to the dialyzed stem cell factor solution as a protective agent, freeze-dried and stored at -80°C to obtain umbilical cord mesenchymal stem cell factor.

[0064] Example 3

[0065] A medicinal umbilical cord mesenchymal stem cell factor comprising epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF).

[0066] The preparation method of the above-mentioned medicinal umbilical cord mesenchymal stem cell factor comprises the following steps:

[0067] S0, obtaining the third generation of expanded cells of umbilical cord mesenchymal stem cells:

[0068] M1: Place the umbilical cord tissue block in a sterile culture dish, add a sterile aqueous solution containing 0.15 wt% collagenase II, 0.15 wt% collagenase IV and 0.08 wt% trypsin, shake and digest at 37.5°C for 70 min, add low-glucose DMEM medium containing 12 wt% FBS to terminate digestion, and obtain a cell suspension; filter the cell suspension through an 80 μm cell screen, centrifuge the filtrate at 1200 rpm for 8 min, and collect the cell pellet;

[0069] M2: Resuspend the cell pellet with low-glucose DMEM medium containing 12 wt% platelet lysate, 1.5 wt% GlutaMAX, and 12 ng / mL basic fibroblast growth factor (bFGF), adjust the cell density to 1×10 7 6% CO2, saturated humidity, replace the medium every 3 days, and subculture when the cell confluence reaches 90%;

[0070] M3: Discard the culture medium, rinse the cells with PBS 3 times, add 0.3 wt% trypsin-EDTA digestion solution, incubate at 37.5°C for 2 min, when the cells become round and start to detach, add low-glucose DMEM medium containing 12 wt% FBS to terminate digestion; blow the cells to form a single cell suspension, inoculate in a new culture bottle at a volume ratio of 1:4 for continuous culture, and the culture medium is the same as that used in primary culture, to obtain subculture expanded cells;

[0071] M4: Repeat the M3 step until the third generation of expanded cells is obtained.

[0072] S1, induction culture: take the 3rd generation of expanded cells of umbilical cord mesenchymal stem cells, and inoculate into an induction medium with a cell density of 1 x 10 7 cells / mL; induction culture is performed in an incubator at 37.5°C, 6% CO2, and saturated humidity for 10 days;

[0073] The induction medium is an endothelial cell culture medium (EGM-2) containing 25 wt% FBS, 60 ng / mL epidermal growth factor (EGF), 30 ng / mL vascular endothelial growth factor (VEGF), 60 μg / mL deferoxamine (HIF-1α stabilizer), 60 μg / mL vitamin C (synergistically enhances VEGF secretion), 2 wt% penicillin-streptomycin, 15 ng / mL rapamycin (promotes autophagy through the mTOR pathway and maintains cell viability), 50 μg / mL asiaticoside, 50 μg / mL astragaloside, 50 μg / mL astragalus polysaccharide, and the balance is deionized water.

[0074] S2, purification and extraction: after the induction culture is completed, the cell culture supernatant is collected; the supernatant is centrifuged at 3500 rpm for 20 min to obtain a centrifuged supernatant; the centrifuged supernatant is loaded onto a HiTrap Heparin HP column previously equilibrated with PBS to capture VEGF; then the chromatography column is washed with PBS for 5 column volumes to remove unbound components to obtain a washing liquid A; then 3 column volumes of elution buffer containing 0.1M glycine-HCl pH3.0 are used for elution, and immediately after, 1M Tris-HCl pH9.2 neutralizing solution is used to adjust the pH value of the eluate to 7.5 to obtain a purified liquid B;

[0075] The washing liquid A is loaded onto a Q Sepharose XL column (pH 8.0) previously equilibrated with PBS to capture EGF; then the chromatography column is washed with PBS for 5 column volumes to remove unbound impurities; then 3 column volumes of elution buffer containing 0.1M glycine-HCl pH3.0 are used for elution, and immediately after, 1M Tris-HCl pH9.2 neutralizing solution is used to adjust the pH value of the eluate to 7.5 to obtain a purified liquid C; the purified liquid B and the purified liquid C are combined to obtain a stem cell factor solution.

[0076] S3, post-treatment: the stem cell factor solution is loaded into a dialysis bag with a molecular weight cut-off of 3.5 kDa, and placed in a dialysis solution containing 20 wt% PBS, and dialyzed at 5°C for 24 h, during which the dialysis solution is replaced 4 times to remove small molecule salts and impurities; 5.5 wt% trehalose is added to the dialyzed stem cell factor solution as a protective agent, and after freeze-drying, it is stored at -80°C to obtain umbilical cord mesenchymal stem cell factor.

[0077] In each of the above examples, the material source: the umbilical cord was derived from a maternal ex vivo umbilical cord. Collagenase II was C6885 from Sigma-Aldrich, with an enzyme activity of 500 U / mg. Collagenase IV was C5138 from Sigma-Aldrich, with an enzyme activity of 200 U / mg. Trypsin was T4799 from Sigma-Aldrich, with an enzyme activity of 2500 U / mg. Low glucose DMEM medium was 11885-084 from Gibco. Platelet lysate was 7901 from StemCell Technologies. GlutaMAX was 35050-061 from Gibco. bFGF (basic fibroblast growth factor) was 100-18B from PeproTech, with an activity of 1 x 10 6 U / mg. PBS (phosphate buffered saline) was SH30256.01 from HyClone. Trypsin-EDTA digestion solution was 25200-056 from Gibco, containing 0.25% trypsin and 0.02% EDTA. Endothelial cell medium (EGM-2) with penicillin-streptomycin was CC-3162 from Lonza. Rapamycin was R-5000 from LC Laboratoires, with a purity of 98%. Epidermal growth factor (EGF) was AF-100-15 from PeproTech, with an activity of 1 x 10 6 U / mg. Vascular endothelial growth factor (VEGF) was 100-20 from PeproTech, with an activity of 1 x 10 6 U / mg. Desferrioxamine was D9518 from Sigma-Aldrich, with a purity of 98%. Vitamin C was A5960 from Sigma-Aldrich, with a purity of 99%. Asperosaponin was derived from Nanjing Zelang Medicine Science and Technology Co., Ltd. Astragaloside IV was derived from Shaanxi Pani'er Biological Technology Co., Ltd. Astragalus polysaccharide was derived from Xi'an Tianzheng Pharmaceutical Auxiliary Material Co., Ltd. HiTrap Heparin HP column was 17-0406-01 from GE Healthcare. Q Sepharose XL column (pH 8.0) was 17-5165-01 from GE Healthcare. Dialysis bag (molecular weight cut-off 3.5 kDa) was 132658 from Spectra / Por. Dialysis solution was Dialysis Solutions from Thermo Fisher Scientific. Trehalose was T9531 from Sigma-Aldrich.

[0078] Comparative Example 1

[0079] In the preparation method of the umbilical cord mesenchymal stem cell factor for medicine: no basic fibroblast growth factor (bFGF) is added in the culture medium in M2, M3 and M4; other parameters and methods are the same as in Example 1.

[0080] Comparative Example 2

[0081] In the preparation method of the umbilical cord mesenchymal stem cell factor for medicine: no epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) are added in the induction medium; other parameters and methods are the same as in Example 1.

[0082] Comparative Example 3

[0083] In the preparation method of the umbilical cord mesenchymal stem cell factor for medicine: no deferoxamine is added in the induction medium; other parameters and methods are the same as in Example 1.

[0084] Comparative Example 4

[0085] In the preparation method of the umbilical cord mesenchymal stem cell factor for medicine: no rapamycin and vitamin C are added in the induction medium; other parameters and methods are the same as in Example 1.

[0086] Comparative Example 5

[0087] In the preparation method of the umbilical cord mesenchymal stem cell factor for medicine: no asiaticoside is added in the induction medium; other parameters and methods are the same as in Example 1.

[0088] Comparative Example 6

[0089] In the preparation method of the umbilical cord mesenchymal stem cell factor for medicine: no astragaloside IV and astragaloside IV are added in the induction medium; other parameters and methods are the same as in Example 1.

[0090] Comparative Example 7

[0091] In the preparation method of the umbilical cord mesenchymal stem cell factor for medicine: no asiaticoside, astragaloside IV and astragaloside IV are added in the induction medium; other parameters and methods are the same as in Example 1.

[0092] The products prepared in each of the above examples and comparative examples are subjected to yield detection, epidermal growth factor (EGF) content detection, vascular endothelial growth factor (VEGF) content detection, and product efficacy detection.

[0093] I. Yield detection

[0094] Detection method: gravimetric method. After freeze-drying, the mass of the umbilical cord mesenchymal stem cell factor obtained was accurately weighed using a high-precision electronic balance, and was recorded as M1 (mg). At the same time, the mass of the umbilical cord tissue block used for preparation was recorded as M0 (mg). The yield calculation formula was: yield = (M1 / M0) x 100%. The detection results are shown in Table 1 below.

[0095] II. Epidermal growth factor (EGF) content detection

[0096] Detection method: enzyme-linked immunosorbent assay (ELISA).

[0097] Preparation: purchase an EGF-specific ELISA kit from a regular reagent company, and according to the kit instructions, dilute the capture antibody to the appropriate concentration (1 μg / mL), add 100 μL per well to the 96-well enzyme-labeled plate, and incubate at 4°C overnight to coat the antibody on the surface of the plate.

[0098] Washing and blocking: discard the coating solution, wash with PBST (0.05% Tween 20 in PBS solution) for 3 times, 3 minutes each time, to remove unbound antibodies. Then add 200 μL blocking solution (5% skim milk in PBS solution), incubate at 37°C for 1 hour, and block non-specific binding sites.

[0099] Sample addition reaction: discard the blocking solution and wash with PBST for 3 times again. Add different dilutions of standard samples (concentrations of 0, 10, 25, 50, 100, 200 ng / mL) and the sample to be tested to the plate, 100 μL per well, and incubate at 37°C for 1 hour to allow the EGF in the sample to bind to the capture antibody.

[0100] Detection antibody incubation: after washing, add biotinylated detection antibody (1:1000 dilution), 100 μL per well, and incubate at 37°C for 30 minutes.

[0101] Affinity-HRP incubation: after washing again, add affinity-HRP (1:2000 dilution), 100 μL per well, and incubate at 37°C for 30 minutes.

[0102] Color development termination: after washing, add 100 μL of TMB substrate solution, react in the dark for 15-20 minutes, allow the HRP to catalyze the substrate to develop color. Finally, add 50 μL of stop solution (2M sulfuric acid solution) to terminate the reaction.

[0103] Reading and calculation: measure the absorbance value at 450 nm wavelength on the enzyme-labeled instrument. Draw a standard curve with the standard sample concentration as the abscissa and the absorbance value as the ordinate. Calculate the EGF content (ng / mL) in the sample according to the standard curve. The detection results are shown in Table 1 below.

[0104] III. VEGF content detection

[0105] Detection method: ELISA method, same as EGF content detection method, using VEGF specific ELISA kit.

[0106] Step parameters: except using VEGF specific antibody and standard, other operation steps and parameters are consistent with EGF detection. The detection results are shown in Table 1 below.

[0107] IV. Product use efficacy detection

[0108] Detection method: cell proliferation experiment is used to detect its cell growth promoting efficacy, and human umbilical vein endothelial cells (HUVEC) are selected as experimental cells.

[0109] Cell inoculation: HUVEC cells are adjusted to a density of 5x10 3 cells per hole with endothelial cell culture medium (EGM-2) containing 10% FBS, inoculated into a 96-well plate at 100 μL per well, and cultured in a 37°C, 5% CO2 incubator overnight to allow cell adhesion.

[0110] Sample addition treatment: different dilutions (1:10, 1:50, 1:100) of stem cell factor solutions prepared by each example and comparative example are added, and a control group (only EGM-2 medium is added) is set, and 5 replicate wells are set for each group. Continue to culture for 48 hours.

[0111] CCK-8 detection: before the end of the culture, 10 μL of CCK-8 solution is added to each well, and incubated at 37°C for 2 hours. The absorbance value is measured at 450 nm wavelength on a microplate reader. The cell proliferation rate calculation formula is: cell proliferation rate = [(experimental group OD value-control group OD value) ÷ control group OD value] x 100%. The detection results are shown in Table 1 below.

[0112] Table 1 detection results

[0113] Sample Yield % EGF content ng / mL VEGF content ng / mL Cell proliferation rate % Example 1 0.60 51 32 80.6 Example 2 0.58 48 29 78.3 Example 3 0.63 55 35 85.1 Comparative Example 1 0.47 38 20 61.8 Comparative Example 2 0.30 9 6 32.2 Comparative Example 3 0.52 43 24 68.7 Comparative Example 4 0.54 41 22 65.4 Comparative Example 5 0.53 45 26 70.3 Comparative Example 6 0.51 42 25 66.0 Comparative Example 7 0.46 31 17 55.4

[0114] From the above results, it can be seen that the product yield and purity of examples 1 to 3 are high, and the cell proliferation effect is outstanding.

[0115] Example 1: When no basic fibroblast growth factor (bFGF) is added to the medium in M2, M3, and M4, bFGF is an important growth factor that can bind to receptors on the cell surface, activate a series of intracellular signaling pathways, including the Ras-Raf-MEK-ERK signaling pathway, promote cell cycle progression, and make cells enter the S phase from the G1 phase, thereby promoting cell proliferation. At the same time, bFGF also plays an important role in maintaining stem cell stemness, it can inhibit the differentiation of stem cells and maintain the pluripotency of stem cells. After the absence of bFGF, the cell proliferation and stemness maintenance ability are weakened, the number of cells is reduced, the secretion of stem cell factor is reduced, and then the yield, EGF and VEGF content, and cell proliferation rate and other detection results are affected.

[0116] Example 2: When no epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) are added to the induction medium, the cell's ability to secrete these factors is limited. EGF and VEGF are key regulators of cell growth and differentiation, they can directly act on cell surface receptors, activate intracellular signaling pathways, and promote cell proliferation, migration, and differentiation. Without the addition of these two factors, the cells lack sufficient growth stimulation signals and cannot effectively secrete EGF and VEGF, resulting in very low levels of these two factors in the product. At the same time, due to the lack of EGF and VEGF to promote cell growth, the promotion of HUVEC cell proliferation is weak, and the yield is also reduced due to the lack of key factor induction.

[0117] Example 3: When no deferoxamine is added to the induction medium, deferoxamine as a HIF-1α stabilizer can simulate a hypoxic state in the cell microenvironment. Under normal oxygen conditions, HIF-1α is rapidly degraded, but deferoxamine can inhibit its degradation, allowing HIF-1α to exist stably. Stable HIF-1α can activate a series of gene expressions related to hypoxic adaptation in cells, including the expression of angiogenesis-related factors such as VEGF. Without deferoxamine, the adaptability of cells to hypoxic environment changes, HIF-1α cannot exist stably, VEGF secretion is reduced, and then cell proliferation and yield are affected, resulting in a decrease in various indicators.

[0118] Example 4: When rapamycin and vitamin C are not added to the induction medium, rapamycin promotes autophagy through the mTOR pathway, which is a self-degradation process in cells that can remove damaged organelles and protein aggregates, maintain the stability of the intracellular environment, and ensure that cells are in a healthy metabolic state during the induction culture process, which is conducive to the sustained secretion of stem cell factors. Vitamin C not only participates in the intracellular antioxidant process, but also synergistically enhances VEGF secretion. It can achieve this effect by regulating the intracellular redox state and affecting the activity of VEGF synthesis-related enzymes. Without these two components, cell autophagy cannot proceed normally, metabolic waste accumulates, cell viability decreases, VEGF secretion is affected, and various indicators decrease.

[0119] Example 5: When asiaticoside is not added to the induction medium, asiaticoside contains various bioactive components that can regulate the immune microenvironment of cells, reduce the damage of inflammatory reactions to cells, and promote cell growth and factor secretion by promoting intercellular communication and enhancing cell-cell interactions. Without asiaticoside, the promotion of cell growth and factor secretion is weakened, and various indicators decrease slightly.

[0120] Example 6: When astragaloside IV and astragaloside are not added to the induction medium at the same time, astragaloside IV and astragaloside can regulate cell immunity, enhance the immune function of cells, and make cells better cope with various stimuli in the culture environment; they can also promote cell growth and factor secretion by promoting intercellular communication and regulating intracellular signal transduction pathways. Without these two components, the synergistic promotion of cell growth and factor secretion is lost, and various indicators decrease slightly.

[0121] Example 7: When asiaticoside, astragaloside IV, and astragaloside are not added to the induction medium at the same time, the promotion of cell growth and factor secretion by these components is completely lost. The cell microenvironment and intracellular signaling pathways are greatly affected, and cell growth and factor secretion are significantly inhibited, leading to further decreases in various indicators.

Claims

1. A method for preparing a pharmaceutical umbilical cord mesenchymal stem cell factor, characterized by, Comprising the following steps: S1: take the 3rd generation of umbilical cord mesenchymal stem cells, and inoculate into the induction medium, with a cell density of 1×10 6 cells / mL~1×10 7 cells / mL, and perform induction culture; The induction culture medium is an endothelial cell culture medium containing 20wt%-25wt% FBS, 50ng / mL-60ng / mL epidermal growth factor, 20ng / mL-30ng / mL vascular endothelial growth factor, 50μg / mL-60μg / mL deferoxamine, 50μg / mL-60μg / mL vitamin C, 1wt%-2wt% penicillin-streptomycin, 10ng / mL-15ng / mL rapamycin, 30μg / mL-50μg / mL asperosaponin, 30μg / mL-50μg / mL astragaloside, 30μg / mL-50μg / mL astragalus polysaccharide, and the balance being deionized water; S2: After the induction culture is completed, the cell culture supernatant is collected; the supernatant is centrifuged to obtain a centrifugal supernatant; the centrifugal supernatant is loaded onto a HiTrap Heparin HP column to capture VEGF; then PBS is used for washing to remove unbound components to obtain a washing liquid A; then an elution buffer containing 0.1M glycine-HCl pH2.5-3.0 is used for elution, and immediately a 1M Tris-HCl pH9.0-9.2 neutralizing solution is used to adjust the pH value of the eluate to 7.0-7.5 to obtain a purified liquid B; The washing liquid A is loaded onto a Q Sepharose XL column to capture EGF; then PBS is used for washing to remove unbound impurities; Then an elution buffer containing 0.1M glycine-HCl pH2.5-3.0 is used for elution, and immediately a 1M Tris-HCl pH9.0-9.2 neutralizing solution is used to adjust the pH value of the eluate to 7.0-7.5 to obtain a purified liquid C; the purified liquid B and the purified liquid C are combined to obtain a stem cell factor solution; S3: The stem cell factor solution is loaded into a dialysis bag, placed in a dialysis solution, and dialyzed; trehalose is added to the dialyzed stem cell factor solution as a protective agent, and after freeze-drying, it is stored at -80℃ to obtain umbilical cord mesenchymal stem cell factor; the umbilical cord mesenchymal stem cell factor comprises epidermal growth factor and vascular endothelial growth factor.

2. The method of claim 1, wherein the umbilical cord mesenchymal stem cell factor is prepared for a pharmaceutical use. The yield of the umbilical cord mesenchymal stem cell factor is 0.58% or more.

3. The method for preparing pharmaceutical-grade umbilical cord mesenchymal stem cell factor according to claim 1, characterized in that, In S1, the induction culture is performed in an incubator at 36.5℃-37.5℃, 5%-6% CO2, and saturated humidity for 7d-10d.

4. The method for preparing pharmaceutical-grade umbilical cord mesenchymal stem cell factor according to claim 1, characterized in that, In S2, the supernatant is centrifuged at 3000rpm-3500rpm for 15min-20min to obtain a centrifugal supernatant; the HiTrap Heparin HP column is equilibrated with PBS in advance before loading; the Q Sepharose XL column is equilibrated with PBS in advance before loading; the PBS washing is 3-5 column volumes; the elution is 2-3 column volumes.

5. The method of claim 1, wherein the umbilical cord mesenchymal stem cell factor is prepared for a pharmaceutical use. In S3, the molecular weight cut-off of the dialysis bag is 3.5kDa; the dialysis solution contains 10wt%-20wt% PBS; the dialysis is performed at 4℃-5℃ for 12h-24h, and the dialysis solution is replaced 3-4 times during the period to remove small molecule salts and impurities; the amount of trehalose added is 4.5wt%-5.5wt% of the mass of the stem cell factor solution.

6. The method of claim 1, wherein the umbilical cord mesenchymal stem cell factor is prepared for a pharmaceutical use. In S1, the method for obtaining the third generation of expanded cells of umbilical cord mesenchymal stem cells comprises the following steps: M1: Put the umbilical cord tissue block into a sterile culture dish, add a sterile aqueous solution containing collagenase II, collagenase IV and trypsin, shake and digest for 50-70 min, add low-sugar DMEM medium containing 10-12 wt% FBS to terminate digestion, and obtain a cell suspension; filter the cell suspension through a cell screen, centrifuge the filtrate, and collect the cell precipitate; M2: resuspend the cell pellet with low glucose DMEM medium containing 10wt%-12wt% platelet lysate, 1wt%-1.5wt% Gluta MAX, 10ng / mL-12ng / mL basic fibroblast growth factor, and adjust the cell density to 1x10 6 7 primary culture, and when the cell confluence reaches 80%-90%, subculture.​ M3: Discard the culture medium, rinse the cells with PBS, add 0.25-0.3 wt% trypsin-EDTA digestion solution, incubate at 36.5-37.5°C for 1-2 min, when the cells become round and start to detach, add low-sugar DMEM medium containing 10-12 wt% FBS to terminate digestion; blow the cells to form a single cell suspension, inoculate in a new culture bottle for continuous culture, and obtain the passage expanded cells; M4: Repeat the M3 step until the third generation of expanded cells is obtained.

7. The method of claim 6, wherein the umbilical cord mesenchymal stem cell factor is prepared for a pharmaceutical use. The sterile aqueous solution contains 0.1-0.15 wt% collagenase II, 0.1-0.15 wt% collagenase IV and 0.05-0.08 wt% trypsin; the temperature for shaking and digestion is 36.5-37.5°C; the cell screen is 70-80 μm; the centrifugal speed of the filtrate is 1000-1200 rpm, and the centrifugal time is 5-8 min.

8. The method of claim 6, wherein the umbilical cord mesenchymal stem cell factor is prepared for a pharmaceutical use. The primary culture is cultured in a culture box at 36.5-37.5°C, 5-6% CO2 and saturated humidity, the culture medium is replaced every 3 days, and the cell confluence reaches 80-90%.

9. The method of claim 6, wherein the umbilical cord mesenchymal stem cell factor is prepared for a pharmaceutical use. The cells are rinsed with PBS for 2-3 times; the volume ratio of inoculation is single cell suspension: medium = 1: (3-4), and the medium is the same as that used for primary culture.