A stem cell composition for regulating sub-health state and its preparation method and application

Abstract

The present invention proposes a stem cell composition for regulating sub-health status, and a preparation method and application thereof, which belong to the field of stem cell technology. After ginseng, Panax notoginseng and Angelica sinensis are fermented, filtered, plant exosomes are extracted by solid, and plant saponins are obtained by liquid separation and purification. Fetal bovine serum, dexamethasone, penicillin, streptomycin, plant exosomes, plant saponins, and cell growth promoting factors are added to DMEM culture medium, and the separated stem cells are incubated and freeze-dried to obtain a stem cell composition for regulating sub-health status. The stem cell composition for regulating sub-health status prepared by the present invention is safe to use, has good biocompatibility, can effectively improve human immunity, promote growth and development, anti-tumor, repair cells in the body, etc., has a good regulating effect on people in sub-health status, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cells, and in particular to a stem cell composition for regulating a sub-healthy state, and a preparation method and application thereof. Background Art

[0002] In addition to the healthy state and the diseased state, the human body also has an intermediate state that is neither healthy nor diseased, namely the sub-healthy state. Its main characteristics are various symptoms reflected by the feeling of physical and mental discomfort, such as fatigue, weakness, mood changes, etc., and its condition is difficult to clarify for a long period of time; there are various weakness manifestations caused by tissue structures or physiological function decline that are not suitable for age, microecological imbalance, and pre-morbid physiological and pathological changes of certain diseases. Sub-health is a dynamic process that is constantly changing. If active intervention is not carried out, it will inevitably deteriorate to a disease state.

[0003] There are three main pathological mechanisms of sub-health: ① Changes in neuroendocrine function: Studies have shown that sub-health caused by various adverse factors acting on the body will eventually cause changes in the function of the hypothalamus-pituitary-adrenal axis and induce clinical symptoms. In addition, studies have found that depression can affect the growth and development of nerve cells, which is manifested as a decrease in nerve cell dendrites. Imaging measurements show that the brain volume is reduced. Therefore, changes in neuroendocrine function play a very important role in the development of sub-health. ② Changes in immune system function: Various stress factors can easily affect the body's immune system function, leading to the occurrence of various immune-related diseases. ③ Abnormal changes in gene expression: Under the stimulation of stress factors, normal tissue cell expression will be abnormal. When the body is subjected to adverse stimulation, it can produce heat shock protein (HSP), which has the function of preventing cell apoptosis and improving the body's tolerance. The stress state will also inhibit the expression of plasminogen activator inhibitor, leading to thrombosis. Under the action of stress factors, the tumor suppressor gene P53 mutates, promoting carcinogenesis.

[0004] Stem cells have the functions of homing, multidirectional differentiation, self-proliferation and paracrine. After reaching organs throughout the body with the blood, they can recognize the signals of inflammatory factors in the body, automatically home to the tissues that need to be repaired, amplify in large quantities and then differentiate into damaged tissue cells, replace damaged cells, and re-stimulate organ function. At the same time, they can release a large number of cytokines, change the cell microenvironment, promote the repair of damaged aging cells and activate surrounding cells, restore body functions and thus achieve the effect of improving sub-health. Although the activity state of stem cells has a very significant effect on its effect of alleviating sub-health, it is difficult to detect and reinfuse cell preparations in the optimal action period and reinfuse them into the body due to the limitations of existing detection technologies and methods, long infusion time and distance, and cell preparations are in vitro active preparations, and their timeliness and activity rate have a greater impact on their functions. How to further improve the effect of stem cells is an urgent problem to be solved in this field. Summary of the invention

[0005] The purpose of the present invention is to propose a stem cell composition for regulating sub-health status and its preparation method and application. It is safe to use, has good biocompatibility, can effectively improve human immunity, promote growth and development, fight tumors, repair cells in the body, etc. It has a good regulating effect on people in sub-health status and has broad application prospects.

[0006] The technical solution of the present invention is achieved in this way:

[0007] The present invention provides a preparation method of a stem cell composition for regulating a sub-healthy state. The method comprises the following steps: fermenting ginseng, notoginseng and angelica, filtering, extracting plant exosomes as solids, separating and purifying the plant saponins as liquids, adding the plant exosomes, plant saponins and cell growth promoting factors into a DMEM culture medium, incubating the separated stem cells, and freeze-drying the stem cells to obtain the stem cell composition for regulating a sub-healthy state.

[0008] As a further improvement of the present invention, the following steps are included:

[0009] S1. Fermentation: Slice ginseng, Panax notoginseng and angelica, mix, add to water, inoculate fermentation bacteria, ferment, filter, retain solids, concentrate the filtrate to obtain a fermentation broth;

[0010] S2. Extraction of exosomes: adding the solid in step S1 to PBS solution, homogenizing, filtering, adding protease inhibitors, adjusting the pH value of the solution, fractionating centrifuging, collecting the supernatant, ultra-high speed centrifuging, resuspending the solid, vortexing and precipitating, adding the precipitate to a mixed solution of sucrose and trehalose, ultra-high speed centrifuging, resuspending the solid, vortexing and precipitating, washing, and freeze-drying to obtain plant exosomes;

[0011] S3 saponin purification: The fermentation broth in step S1 was purified by a macroporous resin and freeze-dried to obtain plant saponins;

[0012] S4. Preparation of culture medium: adding fetal bovine serum, dexamethasone, penicillin, streptomycin, plant exosomes, plant saponin, and cell growth promoting factors to DMEM culture medium to prepare a culture medium;

[0013] S5. Preparation of a stem cell composition for regulating sub-health status: The excised umbilical cord tissue is cleaned with tissue protection solution to remove residual fat, connective tissue and blood, and then disinfected with medical alcohol. The tissue is then digested with digestive solution, centrifuged, and the supernatant is discarded. The digested tissue is collected, cleaned, and added to DMEM culture medium for culture. The tissue clumps are removed, and after the cells adhere to the wall and grow to 80-90% fusion, the culture medium is aspirated, the cells are rinsed with D-Hank's solution, digested, added to the culture medium for incubation, and freeze-dried to obtain a stem cell composition for regulating sub-health status.

[0014] As a further improvement of the present invention, the mass ratio of ginseng, Panax notoginseng and Angelica sinensis in step S1 is 1-3:5-7:2-3, and the fermentation bacteria are Bacillus subtilis and Bacillus licheniformis seed liquid, with a bacterial content of 10 8 -10 9 cfu / mL, the inoculation amounts were 1-2v / v% and 2-3v / v%, respectively, and the fermentation conditions were 25-30°C, 100-200r / min, and the fermentation was carried out for 24-36h.

[0015] As a further improvement of the present invention, the pH value of the PBS solution in step S2 is 7.2-7.4, the graded centrifugation is performed at 5000 r / min for 20-30 min, 10000 r / min for 15-20 min, and 15000 r / min for 10-15 min, the ultrahigh-speed centrifugation is performed at 100000 g for 50-70 min, the sucrose concentration in the mixed solution of sucrose and trehalose is 10-20wt%, the concentration of trehalose is 7-12wt%, and the solution used for resuspension is 15-25mmol / L Tris-HCl solution.

[0016] As a further improvement of the present invention, the macroporous resin in step S3 is selected from at least one of D-101, AB-8, and DM-130, and the eluent is a 40-60wt% ethanol aqueous solution.

[0017] As a further improvement of the present invention, in the culture medium in step S4, the content of fetal bovine serum is 10-20wt%, the content of dexamethasone is 5-15nmol / L, the content of penicillin is 100-120U / mL, the content of streptomycin is 100-120μg / mL, the content of plant exosomes is 0.5-2.5mg / mL, the content of plant saponin is 0.2-0.5mg / L, and the content of cell growth promoting factor is 20-50μg / mL; the cell growth promoting factor is interleukin-6 and vascular endothelial growth factor, and the mass ratio is 10:3-7.

[0018] As a further improvement of the present invention, the digestion solution in step S5 is a PBS buffer solution with a pH of 7.5-7.7 containing 1-2wt% trypsin and 0.5-1wt% alkaline protease; the culture conditions are 36-38°C, CO 2 The concentration is 3-6 v / v%, and the culture is 2-3 weeks; the incubation temperature is 36-38° C., and the time is 30-60 min.

[0019] The present invention further protects a stem cell composition for regulating sub-health status obtained by the above preparation method.

[0020] The present invention further protects the use of the above-mentioned stem cell composition for regulating sub-health in the preparation of a cell preparation for regulating sub-health.

[0021] The present invention has the following beneficial effects:

[0022] The present invention prepares a stem cell composition for regulating subhealth status, including stem cells, plant exosomes, plant saponins, and cell growth promoting factors, wherein the stem cells are umbilical cord-derived stem cells, which regulate the growth and development of immune cells in the microenvironment in the body through intercellular information transmission, and the release of consistent inflammatory factors to achieve immune homeostasis, and the source is wide, the cost is low, and it is easy to obtain. Under the incubation and culture of the culture medium prepared by the present invention, it can promote stem cells to express and secrete umbilical cord-derived stem cell exosomes, antimicrobial peptides, etc., and improve the survival ability of stem cells, improve their anti-pulmonary fibrosis effects, anti-immune rejection and graft-versus-host disease effects, stimulate dormant stem cells in the body, and improve the inflammatory microenvironment in the body.

[0023] Umbilical cord stem cell exosomes can regulate tumor growth, invasion, metastasis and angiogenesis by delivering nucleic acids, proteins, etc. from mother cells to recipient cells, regulate the immune microenvironment, initiate repair and regeneration to restore the function of key cells, and achieve disease treatment.

[0024] The cell growth promoting factors in the culture medium of the present invention include interleukin-6 and vascular endothelial growth factor, which can affect the behavior of stem cells, angiogenesis and proliferation of stem cells, thereby improving the growth and survival activities of stem cells and improving the in vitro survival rate of stem cells.

[0025] Plant exosomes and plant saponins are added to the culture medium and the stem cell composition for regulating sub-health status of the present invention, both of which are derived from a fermented composition of ginseng, Panax notoginseng and Angelica sinensis. After fermentation, the common saponins therein are promoted to be converted into rare saponins, which greatly improves the physiological activity of the prepared plant saponins, and improves its regulatory activity on the components of the signal pathways related to inflammation, oxidative stress, angiogenesis and cancer metastasis. At the same time, it can also promote cell growth and stimulate stem cells to secrete highly active exosomes.

[0026] Plant exosomes can induce apoptosis through different mechanisms, reduce the proliferation of cancer cells, and prevent the angiogenesis process, thereby preventing cancer metastasis, thereby controlling various types of cancers such as liver, skin, breast, colon and lung, thus playing a good role in regulating sub-health status. At the same time, plant exosomes also promote the secretion of exosomes by umbilical cord-derived stem cells, thereby greatly improving the product's activity in improving immunity.

[0027] The stem cell composition for regulating sub-health status prepared by the present invention is safe to use, has good biocompatibility, can effectively improve human immunity, promote growth and development, fight tumors, repair cells in the body, etc. It has a good regulating effect on people in sub-health status and has broad application prospects. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Trypsin, 200,000 U / g, and alkaline protease, 200,000 U / g, were purchased from Xiasheng (Beijing) Enzyme Biotechnology Development Co., Ltd.

[0030] Protease inhibitors were cocktails without EDTA, mixed with AEBSF, Aprotinin, Leupeptin, Bestatin, E-64, and Pepstatin-A, purchased from AbMole China;

[0031] Bacillus subtilis, 20 billion cfu / g, and Bacillus licheniformis, 20 billion cfu / g, were purchased from Shandong Xinxiong Biotechnology Co., Ltd.

[0032] The preparation method of Bacillus subtilis and Bacillus licheniformis seed solution is as follows: the strains are inoculated into a slant culture medium, cultured at 30°C, 100 r / min, for 24 hours, and the strain seed solution is prepared, with a bacterial content of 10 8 -10 9 cfu / mL. Example 1

[0033] This embodiment provides a stem cell composition for regulating sub-health status, which specifically includes the following steps:

[0034] S1. Fermentation: 1g ginseng, 5g Panax notoginseng, 2g angelica slices were mixed and added to 500mL water, sterilized, inoculated with Bacillus subtilis and Bacillus licheniformis seed liquid, the inoculation amount was 1v / v% and 2v / v%, respectively, at 25°C, 100r / min, fermented for 24h, filtered, the solid was retained, and the filtrate was concentrated to half of the original volume to obtain a fermentation liquid;

[0035] S2. Extraction of exosomes: The solid in step S1 is added to a PBS solution with a pH value of 7.2, homogenized, filtered, and a protease inhibitor accounting for 1 wt% of the system weight is added to adjust the solution pH to 7, centrifuged at 5000 r / min for 20 min, 10000 r / min for 15 min, and 15000 r / min for 10 min, respectively, the supernatant is collected, and centrifuged at 100000 g for 50 min. The solid is resuspended in a 15 mmol / L Tris-HCl solution, vortexed and precipitated, and the precipitate is added to a mixed solution of sucrose and trehalose, wherein the sucrose concentration in the mixed solution of sucrose and trehalose is 10 wt%, and the trehalose concentration is 7 wt%, and centrifuged at 100000 g for 50 min. The solid is resuspended in a 15 mmol / L Tris-HCl solution, vortexed and precipitated, washed, and freeze-dried to obtain plant exosomes;

[0036] S3 saponin purification: The fermentation broth in step S1 was purified by D-101 macroporous resin, the eluent was 40wt% ethanol aqueous solution, the eluate was collected and freeze-dried to obtain plant saponins;

[0037] S4. Preparation of culture medium: adding fetal bovine serum, dexamethasone, penicillin, streptomycin, plant exosomes, plant saponin, and cell growth promoting factors to DMEM culture medium to prepare a culture medium;

[0038] In the culture medium, the content of fetal bovine serum is 10wt%, the content of dexamethasone is 5nmol / L, the content of penicillin is 100U / mL, the content of streptomycin is 100μg / mL, the content of plant exosomes is 0.5mg / mL, the content of plant saponin is 0.2mg / L, and the content of cell growth promoting factor is 20μg / mL;

[0039] The cell growth promoting factors are interleukin-6 and vascular endothelial growth factor, with a mass ratio of 10:3;

[0040] S5. Preparation of stem cell composition for regulating sub-health status: The excised umbilical cord tissue was washed with tissue protection solution to remove residual fat, connective tissue and blood, and then disinfected with medical alcohol. The tissue was digested with digestive solution for 30 minutes, centrifuged at 5000r / min for 30 minutes, and the supernatant was discarded. The digested tissue was collected, washed, added to DMEM culture medium, and incubated at 36°C, CO 2 The concentration is 3v / v%, cultured for 2 weeks, the tissue patch is removed, and after the cells adhere to the wall and grow to 80-90% confluence, the culture medium is aspirated, washed with D-Hank's solution, digested with 2wt% trypsin for 30 minutes, added to the culture medium, incubated at 36°C for 30 minutes, and freeze-dried to obtain a stem cell composition for regulating sub-health status;

[0041] The digestion solution is a PBS buffer solution with a pH of 7.5 and containing 1 wt % trypsin and 0.5 wt % alkaline protease. Example 2

[0042] This embodiment provides a stem cell composition for regulating sub-health status, which specifically includes the following steps:

[0043] S1. Fermentation: 3g ginseng, 7g Panax notoginseng, and 3g angelica slices were mixed and added to 500mL of water, sterilized, and inoculated with Bacillus subtilis and Bacillus licheniformis seed liquids at inoculation rates of 2v / v% and 3v / v%, respectively, at 30°C, 200r / min, and fermented for 36h, filtered, the solids were retained, and the filtrate was concentrated to half of the original volume to obtain a fermentation broth;

[0044] S2. Extraction of exosomes: The solid in step S1 is added to a PBS solution with a pH value of 7.4, homogenized, filtered, and a protease inhibitor accounting for 2 wt% of the system weight is added to adjust the pH value of the solution. The solution is centrifuged at 5000 r / min for 30 min, 10000 r / min for 20 min, and 15000 r / min for 15 min, and the supernatant is collected. The solution is centrifuged at 100000 g for 70 min, and the solid is resuspended in a 25 mmol / L Tris-HCl solution, vortexed and precipitated. The precipitate is added to a mixed solution of sucrose and trehalose, wherein the sucrose concentration in the mixed solution of sucrose and trehalose is 20 wt%, and the trehalose concentration is 12 wt%. The solution is centrifuged at 100000 g for 70 min, and the solid is resuspended in a 25 mmol / L Tris-HCl solution, vortexed and precipitated, washed, and freeze-dried to obtain plant exosomes.

[0045] S3 saponin purification: The fermentation broth in step S1 was purified by AB-8 macroporous resin, the eluent was 60wt% ethanol aqueous solution, the eluate was collected and freeze-dried to obtain plant saponins;

[0046] S4. Preparation of culture medium: adding fetal bovine serum, dexamethasone, penicillin, streptomycin, plant exosomes, plant saponin, and cell growth promoting factors to DMEM culture medium to prepare a culture medium;

[0047] In the culture medium, the content of fetal bovine serum is 20wt%, the content of dexamethasone is 15nmol / L, the content of penicillin is 120U / mL, the content of streptomycin is 120μg / mL, the content of plant exosomes is 2.5mg / mL, the content of plant saponin is 0.5mg / L, and the content of cell growth promoting factor is 50μg / mL;

[0048] The cell growth promoting factors are interleukin-6 and vascular endothelial growth factor, with a mass ratio of 10:7;

[0049] S5. Preparation of stem cell composition for regulating sub-health status: The excised umbilical cord tissue was washed with tissue protection solution to remove residual fat, connective tissue and blood, and then disinfected with medical alcohol. The tissue was digested with digestive solution for 30 minutes, centrifuged at 5000r / min for 30 minutes, and the supernatant was discarded. The digested tissue was collected, washed, added to DMEM culture medium, and incubated at 38°C, CO 2 The concentration is 6v / v%, and the cells are cultured for 3 weeks. The tissue patches are removed, and after the cells adhere to the wall and grow to 80-90% confluence, the culture medium is aspirated, washed with D-Hank's solution, digested with 3wt% trypsin for 30 minutes, added to the culture medium, incubated at 38°C for 60 minutes, and freeze-dried to obtain a stem cell composition for regulating sub-health status;

[0050] The digestion solution is a PBS buffer solution with a pH of 7.7 and containing 2 wt % trypsin and 1 wt % alkaline protease. Example 3

[0051] This embodiment provides a stem cell composition for regulating sub-health status, which specifically includes the following steps:

[0052] S1. Fermentation: 2g ginseng, 6g Panax notoginseng, 2.5g angelica slices were mixed and added to 500mL water, sterilized, inoculated with Bacillus subtilis and Bacillus licheniformis seed liquid, the inoculation amount was 1.5v / v% and 2.5v / v%, respectively, at 27°C, 150r / min, fermented for 30h, filtered, the solid was retained, and the filtrate was concentrated to half of the original volume to obtain a fermentation liquid;

[0053] S2. Extraction of exosomes: The solid in step S1 is added to a PBS solution with a pH value of 7.3, homogenized, filtered, and a protease inhibitor accounting for 1.5 wt% of the system weight is added to adjust the pH value of the solution. The solution is centrifuged at 5000 r / min for 25 min, 10000 r / min for 20 min, and 15000 r / min for 12 min, and the supernatant is collected. The solution is centrifuged at 100000 g for 60 min, and the solid is resuspended in a 20 mmol / L Tris-HCl solution, vortexed and precipitated. The precipitate is added to a mixed solution of sucrose and trehalose, wherein the sucrose concentration in the mixed solution of sucrose and trehalose is 15 wt%, and the trehalose concentration is 10 wt%. The solution is centrifuged at 100000 g for 60 min, and the solid is resuspended in a 20 mmol / L Tris-HCl solution, vortexed and precipitated, washed, and freeze-dried to obtain plant exosomes.

[0054] S3 saponin purification: The fermentation broth in step S1 was purified by AB-8 macroporous resin, the eluent was 50wt% aqueous ethanol solution, the eluate was collected and freeze-dried to obtain plant saponins;

[0055] S4. Preparation of culture medium: adding fetal bovine serum, dexamethasone, penicillin, streptomycin, plant exosomes, plant saponin, and cell growth promoting factors to DMEM culture medium to prepare a culture medium;

[0056] In the culture medium, the content of fetal bovine serum is 15wt%, the content of dexamethasone is 10nmol / L, the content of penicillin is 110U / mL, the content of streptomycin is 110μg / mL, the content of plant exosomes is 1.5mg / mL, the content of plant saponin is 0.35mg / L, and the content of cell growth promoting factor is 35μg / mL;

[0057] The cell growth promoting factors are interleukin-6 and vascular endothelial growth factor, with a mass ratio of 10:5;

[0058] S5. Preparation of stem cell composition for regulating sub-health status: The excised umbilical cord tissue was washed with tissue protection solution to remove residual fat, connective tissue and blood, and then disinfected with medical alcohol. The tissue was digested with digestive solution for 30 minutes, centrifuged at 5000r / min for 30 minutes, and the supernatant was discarded. The digested tissue was collected, washed, added to DMEM culture medium, and incubated at 37°C, CO 2 The concentration was 5v / v%, and the cells were cultured for 3 weeks, and the tissue blocks were removed. After the cells adhered to the wall and grew to 80-90% confluence, the culture medium was aspirated, and the cells were washed with D-Hank's solution, digested with 2.5wt% trypsin for 30 minutes, added to the culture medium, incubated at 37°C for 45 minutes, and freeze-dried to obtain a stem cell composition for regulating sub-health status;

[0059] The digestion solution is a PBS buffer solution with a pH of 7.6 and containing 1.5 wt % trypsin and 0.7 wt % alkaline protease. Example 4

[0060] Compared with Example 3, the difference is that the cell growth promoting factor is replaced by interleukin-6. Example 5

[0061] Compared with Example 3, the difference is that the cell growth promoting factor is replaced by vascular endothelial growth factor.

[0062] Comparative Example 1

[0063] Compared with Example 3, the difference is that ginseng is not added in step S1.

[0064] The details are as follows:

[0065] S1. Fermentation: Slice 6g of Panax notoginseng and 2.5g of Angelica sinensis, mix them, add them to 500mL of water, sterilize them, inoculate them with seed liquid of Bacillus subtilis and Bacillus licheniformis at inoculation amounts of 1.5v / v% and 2.5v / v%, respectively, culture them at 27℃, 150r / min, ferment them for 30h, filter them, retain the solids, and concentrate the filtrate to half of the original volume to obtain the fermentation liquid.

[0066] Comparative Example 2

[0067] Compared with Example 3, the difference is that Panax notoginseng is not added in step S1.

[0068] The details are as follows:

[0069] S1. Fermentation: Slice 2g of ginseng and 2.5g of angelica, mix, add to 500mL of water, sterilize, inoculate with Bacillus subtilis and Bacillus licheniformis seed liquid, the inoculation amount is 1.5v / v% and 2.5v / v%, respectively, ferment and culture for 30h at 27℃, 150r / min, filter, retain the solid, and concentrate the filtrate to half of the original volume to obtain the fermentation liquid.

[0070] Comparative Example 3

[0071] Compared with Example 3, the difference is that Angelica sinensis is not added in step S1.

[0072] The details are as follows:

[0073] S1. Fermentation: Mix 2g ginseng and 6g Panax notoginseng slices, add to 500mL water, sterilize, inoculate with Bacillus subtilis and Bacillus licheniformis seed liquid, the inoculation amount is 1.5v / v% and 2.5v / v%, respectively, at 27℃, 150r / min, ferment for 30h, filter, retain the solid, and concentrate the filtrate to half of the original volume to obtain the fermentation liquid.

[0074] Comparative Example 4

[0075] Compared with Example 3, the difference is that plant exosomes are not added in step S4.

[0076] The details are as follows:

[0077] S4. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, plant saponin, and cell growth promoting factor were added to DMEM medium to prepare a culture medium;

[0078] In the culture medium, the content of fetal bovine serum is 15wt%, the content of dexamethasone is 10nmol / L, the content of penicillin is 110U / mL, the content of streptomycin is 110μg / mL, the content of plant saponin is 0.35mg / L, and the content of cell growth promoting factor is 35μg / mL;

[0079] The cell growth promoting factors are interleukin-6 and vascular endothelial growth factor, with a mass ratio of 10:5.

[0080] Comparative Example 5

[0081] Compared with Example 3, the difference is that plant saponin is not added in step S4.

[0082] The details are as follows:

[0083] S4. Preparation of culture medium: adding fetal bovine serum, dexamethasone, penicillin, streptomycin, plant exosomes, and cell growth promoting factors to DMEM culture medium to prepare a culture medium;

[0084] In the culture medium, the content of fetal bovine serum is 15wt%, the content of dexamethasone is 10nmol / L, the content of penicillin is 110U / mL, the content of streptomycin is 110μg / mL, the content of plant exosomes is 1.5mg / mL, and the content of cell growth promoting factor is 35μg / mL;

[0085] The cell growth promoting factors are interleukin-6 and vascular endothelial growth factor, with a mass ratio of 10:5.

[0086] Comparative Example 6

[0087] Compared with Example 3, the difference is that no cell growth promoting factor is added in step S4.

[0088] The details are as follows:

[0089] S4. Preparation of culture medium: adding fetal bovine serum, dexamethasone, penicillin, streptomycin, plant exosomes, and plant saponin to DMEM culture medium to prepare a culture medium;

[0090] In the culture medium, the content of fetal bovine serum is 15wt%, the content of dexamethasone is 10nmol / L, the content of penicillin is 110U / mL, the content of streptomycin is 110μg / mL, the content of plant exosomes is 1.5mg / mL, and the content of plant saponin is 0.35mg / L.

[0091] Test Example 1: Experiment on the determination of RAW264.7 cell viability

[0092] RAW264.7 cells in the logarithmic growth phase were collected and cultured at a density of 5 × 10 4 100 μg / ml were inoculated in a 96-well cell culture plate and cultured in a constant temperature incubator for 24 hours; experimental groups were set up: blank control group; negative control group (20 mmol / L Tris-HCl); positive control group (1 μg / L LPS); drug administration group (0.5 mg / mL of the stem cell composition for regulating subhealth status prepared in Examples 1-5 and Comparative Examples 1-6), 6 replicate wells were set up in each group, and after 24 hours, CCK-8 was added under light-proof conditions and incubated at 37°C for 30 minutes; the OD value of each well was detected at a wavelength of 450 nm. The cell survival rate (%) was calculated.

[0093] The results are shown in Table 1.

[0094] Table 1

[0095]

[0096] It can be seen from the above table that the stem cell compositions for regulating sub-health status prepared in Examples 1-3 of the present invention have low cytotoxicity.

[0097] Test Example 2

[0098] After 1 week of adaptive feeding, nude mice were implanted with SMMC-7721 cells in the axilla. 7 The mice were randomly divided into groups of 10 when the tumor grew to a diameter of 2-3 mm, Examples 1-5 and Comparative Examples 1-6, and a model group. The prepared stem cell composition for regulating sub-health was prepared into a 1 mg / mL suspension with physiological saline, and 2 mL / mouse was administered twice a week. The model group was given an equal amount of physiological saline, both administered via the tail vein. The experimental period was 2 weeks, and the tumor weight inhibition rate (%) was calculated.

[0099] The results are shown in Table 2.

[0100] Table 2

[0101]

[0102] It can be seen from the above table that the stem cell compositions for regulating sub-health status prepared in Examples 1-3 of the present invention have good anti-tumor effects.

[0103] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a stem cell composition for regulating sub-health status, characterized in that: The following steps are involved: S1. Fermentation: Slice ginseng, Panax notoginseng and angelica, mix, add to water, inoculate with fermentation bacteria, ferment and culture, filter, retain the solid, concentrate the filtrate to obtain a fermentation broth; S2. Extraction of exosomes: adding the solid in step S1 to PBS solution, homogenizing, filtering, adding protease inhibitors, adjusting the pH value of the solution, fractionating centrifuging, collecting the supernatant, ultra-high speed centrifuging, resuspending the solid, vortexing and precipitating, adding the precipitate to a mixed solution of sucrose and trehalose, ultra-high speed centrifuging, resuspending the solid, vortexing and precipitating, washing, and freeze-drying to obtain plant exosomes; S3 saponin purification: The fermentation broth in step S1 was purified by a macroporous resin and freeze-dried to obtain plant saponins; S4. Preparation of culture medium: adding fetal bovine serum, dexamethasone, penicillin, streptomycin, plant exosomes, plant saponins, and cell growth promoting factors to DMEM culture medium to prepare a culture medium; in the culture medium, the content of fetal bovine serum is 10-20wt%, the content of dexamethasone is 5-15nmol / L, the content of penicillin is 100-120U / mL, the content of streptomycin is 100-120μg / mL, the content of plant exosomes is 0.5-2.5mg / mL, the content of plant saponins is 0.2-0.5mg / L, and the content of cell growth promoting factors is 20-50μg / mL; the cell growth promoting factors are interleukin-6 and vascular endothelial growth factor, and the mass ratio is 10:3-7; S5. Preparation of a stem cell composition for regulating sub-health status: The excised umbilical cord tissue is cleaned with tissue protection solution to remove residual fat, connective tissue and blood, and then disinfected with medical alcohol. The tissue is digested with digestive solution, centrifuged, and the supernatant is discarded. The digested tissue is collected, cleaned, and cultured in DMEM culture medium. Tissue clumps are removed, and after the cells adhere to the wall and grow to 80-90% fusion, the culture medium is aspirated, the cells are rinsed with D-Hank's solution, digested, added to the culture medium prepared in step S4 for incubation, and freeze-dried to obtain a stem cell composition for regulating sub-health status.

2. The preparation method according to claim 1, characterized in that: In step S1, the mass ratio of ginseng, Panax notoginseng and angelica is 1-3:5-7:2-3, and the fermentation bacteria are Bacillus subtilis and Bacillus licheniformis seed liquid, with a bacterial content of 10 8 -10 9 cfu / mL, the inoculation amounts were 1-2v / v% and 2-3v / v%, respectively, and the fermentation conditions were 25-30°C, 100-200r / min, and the fermentation was carried out for 24-36h.

3. The preparation method according to claim 1, characterized in that: The pH value of the PBS solution in step S2 is 7.2-7.4, the graded centrifugation is performed at 5000 r / min for 20-30 min, 10000 r / min for 15-20 min, and 15000 r / min for 10-15 min, the ultra-high speed centrifugation is performed at 100000 g for 50-70 min, the sucrose concentration in the mixed solution of sucrose and trehalose is 10-20wt%, the trehalose concentration is 7-12wt%, and the solution used for resuspension is 15-25mmol / L Tris-HCl solution.

4. The preparation method according to claim 1, characterized in that: In step S3, the macroporous resin is selected from at least one of D-101, AB-8, and DM-130, and the eluent used for purification is a 40-60wt% ethanol aqueous solution.

5. The preparation method according to claim 1, characterized in that: The digestion solution in step S5 is a PBS buffer with a pH of 7.5-7.7 containing 1-2wt% trypsin and 0.5-1wt% alkaline protease; the culture conditions are 36-38°C, CO2 concentration is 3-6v / v%, and the culture is 2-3 weeks; the incubation temperature is 36-38°C, and the time is 30-60min.

6. A stem cell composition for regulating sub-health status obtained by the preparation method according to any one of claims 1 to 5.

7. Use of the stem cell composition for regulating sub-health state as claimed in claim 6 in preparing a cell preparation for regulating sub-health state.

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