A preparation method and application of umbilical cord mesenchymal stem cells

Abstract

The present invention provides a preparation method and application of umbilical cord mesenchymal stem cells, belonging to the field of biomedical technology. By using the conditioned medium of umbilical cord blood venous endothelial cells secreting exosomes for 48 hours as the basic component, compounding the serum "containing compound Chinese medicine ingredients" as the culture system of umbilical cord mesenchymal stem cells, and compounding hypoxia culture, the obtained mesenchymal stem cells are used to treat post-stroke cognitive impairment (PSCI). The umbilical cord mesenchymal stem cells of the present invention can penetrate the brain-blood barrier and improve the ischemia and hypoxia of tissues in the cerebral infarction area, and also provide a new way of synergistic treatment of PSCI biological treatment and traditional Chinese medicine treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a preparation method of umbilical cord mesenchymal stem cells and applications thereof. Background Art

[0002] Human umbilical cord derived stem cells (UCSCs) are a type of stem cell with multidirectional differentiation potential that has been isolated from human umbilical cord tissue in recent years. UCSCs can not only differentiate into a variety of cells such as fat, cartilage, and cardiomyocytes, but also secrete a variety of biologically active cytokines such as angiogenesis factors and apoptosis factors. In addition, the umbilical cord belongs to perinatal tissue, and UCSCs have the advantages of easy access, large quantity, and no side effects on the donor. It has been widely used in the field of tissue engineering and regenerative medicine. Research results show that mesenchymal stem cells (MSCs) MSCs can not only differentiate into new neurons to repair brain tissue after stroke, but also regulate the microenvironment of neural tissue, reduce inflammatory response in the stroke area, promote neurovascular repair, angiogenesis, neurogenesis, etc., by reducing the area of ​​infarction and reducing the damage to the blood-brain barrier.

[0003] At present, most stem cells cultured in vitro are carried out under 21% normoxic conditions, while the oxygen content of normal human tissues is 3% to 10%, the oxygen content of organs is 1% to 5%, the oxygen content of the bone marrow cavity is 1% to 7%, the oxygen content of arterial blood is 12%, and the oxygen content of venous blood is 5%. Therefore, the oxygen environment of traditional mesenchymal stem cell culture in vitro cannot truly simulate the oxygen environment of stem cells in vivo. However, a large number of studies have shown that the survival rate of mesenchymal stem cells is significantly reduced after transplantation. Within 24 hours after transplantation, more than 90% of stem cells undergo necrosis or apoptosis, which seriously affects the application efficacy of stem cells. How to solve the survival of mesenchymal stem cell transplantation has become the primary difficulty in disease treatment.

[0004] Post-stroke cognitive impairment (PSCI) refers to a clinical syndrome characterized by cognitive impairment that occurs after a stroke event and persists for 6 months. PSCI can be divided into post-stroke cognitive impairment nodementia (PSCIND) and post-stroke dementia (PSD) according to the severity of cognitive impairment. Both have at least one cognitive domain impaired, with the difference being that the living and working abilities of PSD patients are severely impaired, while those of PSCIND patients may be completely normal or slightly impaired. With the increasing aging of the Chinese population, the incidence of PSCI is as high as 53.1%. At the same time, the disability rate of PSCI increases, the quality of life, self-care ability, and mental health of patients decline significantly, and the care pressure of family members and the burden of social disease treatment increase sharply, and corresponding treatment methods are urgently needed. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for preparing umbilical cord mesenchymal stem cells and its application. To achieve the above purpose, the technical solution adopted by the present invention is:

[0006] In a first aspect, the present invention provides a drug-containing serum culture medium, comprising a conditioned medium and a drug-containing serum; the drug-containing serum is the serum of rats treated by intragastric administration with a Chinese medicine formula extract; the Chinese medicine formula comprises the following ingredients in parts by weight: 20-40 parts of Astragalus, 5-7 parts of Ginseng, 10-20 parts of Polygonum multiflorum, 10-20 parts of Angelica sinensis, 5-7 parts of Gastrodia elata, 5-7 parts of Polygala tenuifolia, 10-20 parts of Morinda officinalis, 5-7 parts of Pinellia ternata, and 5-7 parts of Acorus calamus.

[0007] The Chinese medicine formula of the present invention can effectively treat cognitive impairment after stroke through the reasonable compatibility of various Chinese medicines. Among them, ginseng (raw sun-dried ginseng) is used as the "main medicine" in the prescription, which plays a role in replenishing vital energy; astragalus, angelica, processed Polygonum multiflorum, and Morinda officinalis have the function of replenishing qi and blood, and are beneficial to the power of essence, as "minister medicine". Astragalus, angelica, Gastrodia elata, Pinellia ternata, Acorus calamus, and Polygala tenuifolia are compatible, which can also play the auxiliary effects of invigorating qi and dredge meridians, replenishing blood and activating blood circulation, and clearing phlegm and unblocking collaterals; Acorus calamus and Polygala tenuifolia have the effects of clearing phlegm and opening the mind, and promoting the flow of spirits. The present invention uses Chinese medicine components and exosomes secreted by venous endothelial cells to "induce" the angiogenic ability of umbilical cord mesenchymal stem cells; the umbilical cord mesenchymal stem cells treated with hypoxia and drug-containing serum have enhanced ability to differentiate into various cells such as fat, cartilage, and cardiomyocytes.

[0008] More preferably, the Chinese medicine formula comprises the following ingredients by weight: 30 parts of Astragalus, 6 parts of Ginseng, 15 parts of Polygonum multiflorum, 15 parts of Angelica sinensis, 6 parts of Gastrodia elata, 6 parts of Polygala tenuifolia, 15 parts of Morinda officinalis, 6 parts of Pinellia ternata, and 6 parts of Acorus calamus.

[0009] As a preferred embodiment of the culture medium of the present invention, the volume ratio of the conditioned culture medium to the drug-containing serum is conditioned culture medium: drug-containing serum = 1: (0.2-0.3).

[0010] As a preferred embodiment of the culture medium of the present invention, the preparation of the Chinese medicine formula extract comprises the following steps: weighing the Chinese medicine formula, adding 1000-1200 mL of water, soaking for 25-30 minutes and then boiling over high heat, then decocting over low heat until the volume is reduced to 500-600 mL and filtering while hot, and concentrating the filtrate to obtain the Chinese medicine formula extract.

[0011] As a preferred embodiment of the culture medium of the present invention, the concentration of the Chinese medicine formula extract is 132.3 mg / mL.

[0012] As a preferred embodiment of the culture medium of the present invention, the preparation of the drug-containing serum comprises the following steps: subjecting rats to intragastric administration with an extract containing a Chinese medicine formula, and then subjecting the rats to blood sampling and purification to obtain the drug-containing serum; the frequency of the intragastric administration is 3 times a day for 7 consecutive days; in the intragastric administration, the amount of the Chinese medicine formula extract is 5 mL / time. The present invention can further increase the concentration of Chinese medicine small molecules in rat serum by optimizing the frequency of the intragastric administration.

[0013] As a preferred embodiment of the culture medium of the present invention, the blood collection process comprises the following steps: 1 hour after the last administration of the drug by gavage, the rats are anesthetized and blood is collected from the abdominal aorta to obtain drug-containing raw blood; after the drug-containing raw blood is allowed to stand at room temperature for 2 hours, it is centrifuged at 3500r / min and 4°C for 10 minutes to separate the serum, which is heat-treated at 56°C for 30 minutes for inactivation, and filtered through a 0.22um filter membrane to obtain drug-containing serum.

[0014] As a preferred embodiment of the culture medium of the present invention, the preparation method of the conditioned medium is as follows: after culturing the P3 generation cells of umbilical vein endothelial cells with complete culture medium for 24-56 hours, the supernatant of the culture system is separated to obtain the conditioned medium. The umbilical vein endothelial cells are subcultured with complete culture medium, and when cultured to 48-56 hours, the umbilical vein endothelial cells will secrete exosomes, and the exosomes are located in the culture supernatant. The exosomes secreted by the umbilical vein endothelial cells contain growth factors, cytokines, proteins, etc. These nutritional factors can promote the growth, proliferation and differentiation of cells and maintain the normal function of cells.

[0015] Therefore, the conditioned medium of umbilical cord blood venous endothelial cells secreting exosomes for 48 hours is used as the basic component, and the serum containing compound Chinese medicine ingredients is used as the cell culture system of UCSCs. The conditions of hypoxia culture can not only provide the nutrients required for mesenchymal stem cell culture, but also simulate the effect of enhanced differentiation ability of mesenchymal stem cells after taking Chinese medicine. The mesenchymal stem cells prepared by this culture method can regulate the density of vascular endothelial cells at multiple levels through their differentiation ability when used in the treatment of cognitive impairment after stroke, and can easily pass through the blood-brain barrier to improve the condition of brain tissue in the cerebral infarction area (ischemic hypoxia area).

[0016] In a second aspect, the present invention provides a method for preparing umbilical cord mesenchymal stem cells, comprising the following steps:

[0017] (1) Primary culture and subculture of umbilical cord mesenchymal stem cells: Isolate the Wharton's jelly in the umbilical cord, separate the umbilical cord mesenchymal stem cells after primary culture, and continue subculture to obtain P3-P5 umbilical cord mesenchymal stem cells;

[0018] (2) Hypoxia treatment with medicated serum: Take the P3-P5 umbilical cord mesenchymal stem cells described in step (1), culture them in a medicated serum medium under hypoxic conditions until the cell fusion rate reaches 90%, collect and resuspend the cells, and obtain umbilical cord mesenchymal stem cells with enhanced differentiation ability. The present invention uses a medicated serum medium to culture umbilical cord mesenchymal stem cells to simulate the effect of inducing the angiogenesis ability of mesenchymal stem cells after taking traditional Chinese medicine.

[0019] As a preferred embodiment of the method of the present invention, in step (1), the primary culture of umbilical cord mesenchymal stem cells specifically comprises the following steps: taking the umbilical cord of a healthy full-term caesarean section baby, cleaning it, peeling off the umbilical cord amniotic membrane, one artery and two veins, and taking out the Wharton's jelly; cutting the Wharton's jelly into pieces of 1-3 cm 3 , resuspended with complete medium and inoculated into T75 culture flask for 2 days, supplemented with complete medium on the second day, and replaced with liquid on the fifth day; the liquid replacement treatment is to resuspend with complete medium and inoculate into T75 culture flask until the cells crawl out, discard the tissue blocks, supplement with complete medium and continue to culture, culture the cells until a larger clone is formed, and obtain P0 generation umbilical cord mesenchymal stem cells that can be passed down. The present invention ensures the purity of cell origin and avoids contamination of vascular endothelial cells by stripping non-target tissues such as amniotic membrane and blood vessels of the umbilical cord. At the same time, the selected complete medium is specially developed for umbilical cord-derived mesenchymal stem cells, and the surface characteristics of mesenchymal stem cells are kept stable after continuous passage.

[0020] As a preferred embodiment of the method of the present invention, in step (1), the subculture of the umbilical cord mesenchymal stem cells specifically includes the following steps: taking the P0 generation umbilical cord mesenchymal stem cells obtained from the primary culture and performing repeated subculture treatment to obtain umbilical cord mesenchymal stem cells expanded to the P3-P5 generation; the subculture treatment sequentially includes the steps of washing, digestion, pipetting and centrifugation, and resuspension culture; when the resuspension culture is carried out until the cells are fused, the digestion, pipetting and centrifugation and resuspension steps are repeated 2-5 times to obtain umbilical cord mesenchymal stem cells expanded to the P2-P5 generation.

[0021] Furthermore, the subculture process specifically comprises the following steps:

[0022] (2a) taking passaged P0 umbilical cord mesenchymal stem cells and washing them 2-3 times with physiological saline;

[0023] (2b) adding 0.25% trypsin to the P0 umbilical cord mesenchymal stem cells washed in step (2a) for digestion; when the intercellular spaces increase, the cytoplasm shrinks, and the long spindle cells become round and brighter as observed under a microscope, adding 10 times the cell volume of complete culture medium to terminate the digestion;

[0024] (2c) repeatedly blowing and beating the umbilical cord mesenchymal stem cells after digestion in step (2b) until all of them fall off into a single cell suspension, and centrifuging the suspension; the centrifugation condition is 1500 rpm, 5 min;

[0025] (2d) Discard the supernatant from step (2c), add complete medium to resuspend the cells, and culture at 1×10 5 The density of cells was 5% CO2, 37℃, saturated humidity environment for umbilical cord mesenchymal stem cell subculture;

[0026] (2e) When the cells grow to confluence, repeat steps (2b) to (2d) 2-5 times as one subculture cycle to expand the cells, thereby obtaining umbilical cord mesenchymal stem cells expanded to P2-P5 generations.

[0027] As a preferred embodiment of the method of the present invention, the primary culture and subculture described in step (1) are both carried out in a T75 culture flask, and the cell culture environment is: 35°C-38°C, 4%-6% CO2, and saturated humidity; the operations in step (1) are all aseptic operations. Preferably, the cell culture environment is 37°C, 5% CO2, and saturated humidity.

[0028] As a preferred embodiment of the method of the present invention, in step (2), the P3-P5 umbilical cord mesenchymal stem cells are cultured at 1×10 5 The cells were inoculated at a density of 100 μg / mL into a medium containing drug serum.

[0029] As a preferred embodiment of the method of the present invention, in step (2), the hypoxic condition is that the cells are cultured at an oxygen content of 1%-3%. More preferably, the oxygen content is 2%.

[0030] Oxygen is a key factor affecting the proliferation of mesenchymal stem cells (MSCs). The oxygen volume fraction of conventional cells cultured in vitro is about 21%, the CO2 volume fraction is about 5%, and the oxygen volume fraction in normal human tissues and interstitial spaces is 3%-9%. Therefore, the present invention improves the proliferation ability of mesenchymal stem cells by simulating the hypoxic cell environment in the human body; on the other hand, culturing mesenchymal stem cells under hypoxic conditions can simultaneously enhance the expression and secretion of mesenchymal stem cell (MSC) trophic factors, including growth factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), transforming growth factor β1 (TGF-β1), insulin-like growth factor 1 (IGF-1), fibroblast growth factor 10 (FGF10) and epidermal growth factor (EGF); cytokines, such as IL-6, IL-8, chemokine ligand 20 (CCL-20) and monocyte chemoattractant protein-1 (MCP-1); and matrix regulators such as angiogenin, metalloproteinase-1 (TIMP-1) and tissue inhibitor of metalloproteinase (MMP).

[0031] The mesenchymal stem cell preparation method of the present invention can improve the survival rate of mesenchymal stem cells cultured in vitro by simulating culture in a hypoxic environment; at the same time, umbilical cord mesenchymal stem cells are cultured with drug-containing serum to induce the differentiation ability (including angiogenesis ability, osteogenic ability, and adipogenesis ability) of the mesenchymal stem cells to be enhanced, further simulating the synergistic effect of mesenchymal stem cells and drug components after human body takes traditional Chinese medicine; the preparation method of the present invention is simple and easy to operate, and has low requirements for instruments and equipment. Ordinary clean benches, low-speed centrifuges and shakers can meet the requirements, and are more conducive to large-scale production and preparation of mesenchymal stem cells used in drug preparation.

[0032] In a third aspect, the present invention provides a method for preparing umbilical cord mesenchymal stem cells and their use in preparing a drug for treating post-stroke cognitive impairment.

[0033] Umbilical cord mesenchymal stem cells can not only be directed to differentiate into various cells such as fat, cartilage, and cardiomyocytes, but also secrete various cytokines with biological activity such as various angiogenesis-promoting factors and apoptosis factors. The umbilical cord mesenchymal stem cells with enhanced differentiation ability prepared by the preparation method of the present invention can differentiate into new neurons to repair brain tissue after stroke, and can also regulate the microenvironment of neural tissue, reduce the inflammatory response in the stroke area, promote neural vascular repair and angiogenesis, neurogenesis, etc., and reduce the infarct area and reduce the damage of the blood-brain barrier.

[0034] As a preferred embodiment of the application of the present invention, the concentration of umbilical cord mesenchymal stem cells in the drug for treating post-stroke cognitive impairment is 1×10 6 cells / mL-1×10 7 cells / mL.

[0035] As a preferred embodiment of the application of the present invention, the drug for treating post-stroke cognitive impairment is administered by injection; the frequency of the injection is 0.1-1.0 mL / time / week, and the administration is performed continuously for 3-5 weeks; the injection site is the lateral ventricle of the brain.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] 1. The present invention provides a method for preparing umbilical cord mesenchymal stem cells. The method is easy to operate and has low requirements for instruments and equipment. Ordinary clean benches, low-speed centrifuges and shakers can meet the requirements, which is more conducive to large-scale production and preparation. At the same time, by stripping non-target tissues such as the amniotic membrane and blood vessels of the umbilical cord, the purity of the cell source is guaranteed and the contamination of vascular endothelial cells is avoided. Using a cell culture system that does not contain animal-derived serum and adding "traditional Chinese medicine ingredients" to culture mesenchymal stem cells under low oxygen conditions can not only provide the nutrients required for mesenchymal stem cell culture, but also simulate the effect of enhancing the differentiation ability of mesenchymal stem cells after the human body takes traditional Chinese medicine, which is induced by the traditional Chinese medicine ingredients.

[0038] 2. The Chinese medicine formula provided by the present invention, wherein ginseng (raw sun-dried ginseng) is used as the "main medicine" in the formula, which plays a role in replenishing vital energy; astragalus, angelica, processed Polygonum multiflorum, Morinda officinalis play the role of replenishing qi and blood, and are beneficial to the power of essence, as "minister medicine". Astragalus, angelica, Gastrodia elata, Pinellia ternata, Acorus calamus, and Polygala tenuifolia are used in combination, which can also play the auxiliary effects of invigorating qi and dredge meridians, replenishing blood and activating blood circulation, and clearing phlegm and unblocking collaterals; Acorus calamus and Polygala tenuifolia have the effects of clearing phlegm and opening the mind. The present invention uses serum containing Chinese medicine ingredients and exosomes secreted by venous endothelial cells to "induce" the angiogenic ability of umbilical cord mesenchymal stem cells.

[0039] 3. In the application provided by the present invention, the umbilical cord mesenchymal stem cells with enhanced differentiation performance after drug-containing hypoxia treatment are used to prepare drugs for treating post-stroke cognitive impairment. The differentiation ability of umbilical cord mesenchymal stem cells is utilized to regulate the microenvironment of neural tissue, so that they can easily pass through the blood-brain barrier and improve the condition of brain tissue in the cerebral infarction area (ischemic and hypoxic area), thereby synergizing with the drug ingredients taken by the patient to treat post-stroke cognitive impairment. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1The flowchart is a method for preparing drug-containing umbilical cord mesenchymal stem cells;

[0041] Figure 2 This is a diagram showing the results of the differentiation function test of drug-containing mesenchymal stem cells;

[0042] Figure 3 The cell morphology of drug-containing mesenchymal stem cells of different generations;

[0043] Figure 4 The figure is the cell viability detection diagram of drug-containing mesenchymal stem cells of different generations;

[0044] Figure 5 This is the cell size diagram of drug-containing mesenchymal stem cells at generation P3;

[0045] Figure 6 This is the cell size diagram of drug-containing mesenchymal stem cells at generation P4;

[0046] Figure 7 This is the cell size diagram of drug-containing mesenchymal stem cells at generation P5;

[0047] Figure 8 This is a graph showing the results of drug-containing mesenchymal stem cells promoting microtubule formation at generations P3-P5. DETAILED DESCRIPTION

[0048] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0049] Example 1 Preparation of drug-containing mesenchymal stem cells

[0050] 1. Primary Culture of Umbilical Cord Stem Cells

[0051] The umbilical cord of a healthy full-term caesarean section baby was taken and sterilely cleaned in a clean bench. The amniotic membrane, one artery and two veins of the umbilical cord were stripped off, and the Wharton's jelly was removed. The Wharton's jelly tissue was cut into pieces with surgical scissors, and the size was 1-3 cm. 3 , resuspended with Youkang complete medium (Q) and inoculated into T75 culture flasks, and transferred to a 5% CO2, 37°C environment for culture. The Youkang complete medium comes from the umbilical cord mesenchymal stem cell serum-free medium set of Youkang Biological, with the item number NC0103.

[0052] Replenish the medium after 2 days, and replace the medium after 5 days. Resuspend the cells with Youkang complete medium (Q) and inoculate them into the original T75 culture flask until the cells crawl out. Discard the tissue pieces and continue to culture with new Youkang complete medium (Q). When the cells form larger clones, they can be passaged.

[0053] 2. Subculture of Umbilical Cord Stem Cells

[0054] Take the P0 cells that can be passaged, discard the original culture medium, add 10 mL of physiological saline to wash, discard the washing solution, and repeat the operation once.

[0055] Add 3 mL of 0.25% trypsin and shake the T75 culture flask left and right to evenly cover the bottom of the dish with 0.25% trypsin; under the microscope, it can be seen that the intercellular space increases and the cytoplasm shrinks; when the long spindle cells become round and bright, immediately add 10 times the volume of Youkang complete medium (Q) to terminate the digestion.

[0056] Repeatedly blow and beat until all cells are detached into a single cell suspension, transfer the suspension to a centrifuge tube, and centrifuge at room temperature at 1500 rpm for 5 minutes.

[0057] Discard the supernatant and add Yokohama complete medium (Q) to resuspend the cells at a density of 1×10 5 The culture flask was placed in a cell culture incubator at 37°C, 5% carbon dioxide and saturated humidity.

[0058] When the cells grow to confluence, repeat the digestion, pipetting and resuspension operations to expand the cells.

[0059] Unless otherwise specified, the human umbilical cord stem cells used in this study were from passages P3 to P5.

[0060] 3. Preparation of Drug-containing Serum

[0061] (1) Preparation of composite water extract: First, take 30 g of Astragalus, 6 g of Ginseng, 15 g of Polygonum multiflorum, 15 g of Angelica sinensis, 6 g of Gastrodia elata, 6 g of Polygala tenuifolia, 15 g of Morinda officinalis, 6 g of Pinellia ternata, and 6 g of Acorus calamus. Place the Chinese medicinal components in a decoction pot, add 1200 mL of water, soak in distilled water for 30 min, boil over high heat, then turn to low heat and continue to decoct until 600 mL remains. Filter while hot, and concentrate the filtrate by rotary evaporation to a concentrated solution with a concentration of 132.3 mg / mL of the synthetic medicinal materials to obtain a composite water extract. The medicinal solution is placed in a 50 mL centrifuge tube and stored in a 4°C refrigerator for later use.

[0062] (2) Preparation of drug-containing serum: Rats were divided into 3 groups and given different doses of the composite water extract according to their body weight: 0.315 mg / g, 0.63 mg / g, and 1.26 mg / g, 3 times a day by gavage, 5 mL each time, for 7 consecutive days. One hour after the last administration, 0.1 chloral hydrate (3 mL / kg) was used for anesthesia, and blood was collected from the abdominal aorta. After standing at room temperature for 2 hours, the blood was centrifuged at 3500 r / min for 10 minutes (4°C) to separate the serum. The drug-containing serum of the same group was combined, heat-treated at 56°C for 30 minutes to inactivate, filtered through a 0.22 um filter membrane, and stored frozen in aliquots.

[0063] 4. Hypoxia treatment of mesenchymal stem cells with drug-containing serum

[0064] (1) Preparation of medium containing drug serum: umbilical cord vein endothelial cells are taken and passaged to P3, and the P3 umbilical cord vein endothelial cells are cultured with Youkang complete medium for 48 hours until the umbilical cord vein endothelial cells secrete exosomes to obtain conditioned medium. Add 20% to 30% of drug-containing serum according to the volume of the conditioned medium to complete the preparation of the drug-containing serum medium;

[0065] (2) P3-P5 umbilical cord mesenchymal stem cells were collected at a density of 1×10 5 The cells were subcultured and inoculated into T75 culture flasks containing medium containing drug serum;

[0066] (3) The cells were transferred to a three-gas incubator (Heal Force, USA) for hypoxic culture. When the oxygen content was 2% and the cell fusion rate was 90%, the cells were collected and a cell suspension was prepared with physiological saline at 1 ml / tube.

[0067] Example 2: Detection of differentiation function of drug-containing mesenchymal stem cells from generations P3 to P5

[0068] The differentiation function of P3-P5 generation drug-containing mesenchymal stem cells was tested, including osteogenic differentiation, adipogenic differentiation, and chondrogenic differentiation. The results are as follows Figure 2 shown.

[0069] 1. Osteogenic differentiation: The osteogenic differentiation medium is DMEM containing 10 mol / L dexamethasone, 10 mmol / L β-glycerol phosphate, 0.05 mmol / L 2-phosphoascorbic acid and 10% fetal bovine serum. 4 / cm 2 The cells were seeded in a 12-well plate, and the osteogenic differentiation medium was replaced when the cell confluence reached 70%-80%. The medium was changed twice a week, and the cells were cultured for about 21 days. Alizarin red staining: PBS was used to rinse once, and the cells were fixed with 95% ethanol at room temperature for 30 minutes, and then 0.2% Alizarin red S was used to stain for 30 minutes. The results were observed under a microscope. Figure 2 (A) is shown. Figure 2 (A) It can be seen that after osteogenic induction, calcium nodules can be seen in P3-P5 cells of UCSCs treated with hypoxia containing medicated serum of the present invention, after staining, indicating that the osteogenic differentiation ability of UCSCs treated with hypoxia containing medicated serum of the present invention is good.

[0070] 2. Adipogenic differentiation: The adipogenic differentiation medium is DMEM containing 10-6 mol / L dexamethasone, 10 mg / L insulin, 0.5 mmol / L isobutylxanthine (IBMX), 200 μmol / L indomethacin and 10% fetal bovine serum.4 / cm 2 Inoculate in a 12-well plate, and when the cell confluence is 70%-80%, replace with adipose-induced differentiation medium. Change the medium twice a week, culture for about 14 days (oil droplets can be seen in the cells under the microscope), and stain with Oil Red O for microscopy: rinse twice with PBS, fix with 95% ethanol at room temperature for 30 minutes, and stain with Oil Red O at room temperature for 30 minutes. Observe under a microscope. The results are as follows: Figure 2 (B) Figure 2 (B) It can be seen that the UCSCs P3-P5 cells treated with hypoxia containing the drug serum of the present invention are induced to adipogenesis and stained to show oil droplets, indicating that the UCSCs treated with hypoxia containing the drug serum of the present invention have good adipogenic differentiation ability.

[0071] 3. Chondrogenic differentiation: Chondrogenic differentiation medium is DMEM medium containing 0.1 μmol / L dexamethasone, 50 mg / L L-ascorbic acid, 1 mmol / L sodium pyruvate, 10 μg / L transforming growth factor β, 50 g / L ITS, and 10% fetal bovine serum. 4 / cm 2 The cells were inoculated into a 12-well plate and replaced with chondrogenic differentiation medium when the cell confluence reached 70%-80%. The medium was changed twice a week. After 14 days of culture, the cells were stained with Alcian blue: washed once with PBS, fixed with 95% ethanol at room temperature for 30 minutes, and stained with 1% Alcian blue for 30 minutes. The results were observed under a microscope. Figure 2 (C)-(D) are shown. Figure 2 (C)-(D) show that the UCSCs P3-P5 cells treated with hypoxia containing the drug-containing serum of the present invention are induced to be cartilage-forming and after staining, calcium nodules can be seen, indicating that the UCSCs treated with hypoxia containing the drug-containing serum of the present invention have good cartilage differentiation ability.

[0072] Example 3 Detection of morphology and surface antigens of drug-containing umbilical cord mesenchymal stem cells

[0073] 1. Cell morphology: Take P3-P5 umbilical cord mesenchymal stem cells and place them under an inverted microscope for observation and photography. Figure 3 As shown in the figure, the cells are uniform in size and spindle-shaped, and appear whirlpool-shaped when 80-90% fused. The cells divide and proliferate quickly and are in good condition.

[0074] 2. Cell viability: Take P3-P5 cells and use trypan blue to test the viability. The results are as follows: Figure 4 As shown; under the trypan blue detection, the cells are all resistant to staining (dead cells are stained blue, and live cells are resistant to staining); this shows that the cells prepared by the method of the present invention are not dead and the cell viability is good.

[0075] 3. Cell size: Take P3-P5 cells to test the cell size distribution. The results are as follows: Figure 5-Figure 7 As shown. Figure 5-Figure 7 From the cell size distribution diagram, we can see that the cell diameter is between 11-23μm, which is consistent with the volume size distribution of mesenchymal stem cells, indicating that the stem cells do not have abnormal phenomena such as volume enlargement.

[0076] From the above pictures, we can see that the umbilical cord mesenchymal stem cells of generations P3 to P5 cultured with medicated serum all grew adherently, with a spindle-shaped cell morphology and a vortex-shaped fusion site. The cell viability was normal and the cell particle size met the standards, indicating that there was no abnormality in the morphology of the stem cells cultured with medicated serum.

[0077] 4. Surface antigen detection: Take the drug-containing serum to treat umbilical cord mesenchymal stem cell P3 cells (sample 1), P4 cells (sample 2), and P5 cells (sample 3) with hypoxia, and make 1×10 6 cells / mL cell suspension, take 5μL of monoclonal antibodies against human CD45, CD14, CD73, CD90, CD105, HLA-DR, CD11b, CD34, add 500μL of cell suspension cultured from the three samples, incubate at room temperature in the dark for 20min, set up blank isotype control at the same time, centrifuge at 1500r / min for 5min, discard the supernatant, wash twice with PBS containing 10% FBS, resuspend with 500μL PBS and test on the machine. The test results are shown in Table 1 below:

[0078] Table 1 Detection of surface antigens of drug-containing mesenchymal stem cells of generation P3-P5

[0079] Surface antigen CD73 CD90 CD105 CD14 CD45 HLA-DR CD11b CD34 P3 cells 99.7％ 97.6％ 98.3％ 0.8％ 1.3％ 0.1％ 0.9％ 0.4％ P4 cells 98.2％ 96.8％ 99.7％ 1.6％ 1.3％ 0.0％ 0.6％ 0.2％ P5 cells 98.8％ 97.2％ 98.1％ 0.8％ 0.7％ 1.3％ 1.4％ 0.2％

[0080] From the above flow cytometry results, we can see that mesenchymal stem cells treated with hypoxia containing drug serum highly expressed CD73, CD90, and CD105, all higher than 95%. They lowly expressed HLA-DR, CD45, CD11b, CD19, and CD34, with expression rates all lower than 2%. This is consistent with the surface antigen characteristics of mesenchymal stem cells, indicating that mesenchymal stem cells treated with hypoxia containing drug serum did not differentiate and still maintained the characteristics of stem cells.

[0081] 5. Test of the ability of drug-containing umbilical cord mesenchymal stem cells to promote microtubule formation: In the microtubule formation experiment, serum-free culture medium (pre-cooled on ice) was used to dilute the matrix gel (BD Company, USA) at a ratio of 1:1. After mixing, 200 μL of the mixture was added to a 24-well plate (all operations must be performed on an ice box), and then flattened and placed at 37°C for 30 minutes. It can be used only after the matrix gel has agglutinated. UCMSCs of P3 to P5 generations (cell number 3×10 4) were placed in the upper layer of the Transwell chamber, and human umbilical vein endothelial cells (HUVECs) were added to the 24-well plate covered with matrix gel. After the two were interacted for 6 hours, photos were taken and analyzed. The results are shown in Figure 8 As shown in the figure, it can be seen that the venous endothelial cells all form a cavity-like hollow structure, indicating that UCSCs cultured with drug-containing serum can promote the formation of microtubules by venous endothelial cells.

[0082] Example 4: Drug-containing umbilical cord mesenchymal stem cell intervention method for post-stroke cognitive impairment

[0083] 1. Post-stroke cognitive impairment (PSCI) rat model

[0084] After 2 weeks of adaptive feeding, all rats were subjected to Morris water maze test 3 days before surgery. Those rats that could not find the platform within 60 seconds were discarded, and it was determined that there was no significant difference in intelligence between rats. They were randomly divided into 3 groups: 10 in the blank group, 10 in the model group, and 10 in the treatment group.

[0085] Modeling method: The VD animal model was established by permanent ligation of bilateral common carotid arteries. Rats were fasted for 12 hours and deprived of water for 4 hours before modeling. They were anesthetized by intraperitoneal injection of 10% chloral hydrate (4 ml / kg) to ensure spontaneous breathing during surgery. The rats were fixed in a supine position, the front of the neck was depilated and disinfected, and the midline of the neck was incised. The bilateral common carotid arteries were isolated and ligated with double silk thread to establish the PSCI rat animal model. Rats in the blank group underwent sham surgery.

[0086] Successful modeling criteria: The time for the PSCI model rats to cross the original platform for the first time (escape latency) was significantly prolonged, and the frequency of crossing the original platform was significantly less than that of the control normal rats.

[0087] 2. Drug-containing umbilical cord mesenchymal stem cell cell intervention test

[0088] A total of 66 SD rats were randomly divided into a normal group, a model group, a low-drug serum cell therapy group (1×10 6 cells, 5×10 6 cells, 1×10 7 cells), and the drug-containing serum cell treatment group (1×10 6 cells, 5×10 6 cells, 1×10 7 cells), high drug-containing serum cell therapy group (1×10 6 cells, 5×10 6 cells, 1×10 7cells). The middle dose is the converted dose of the Chinese medicine formula concentrate for normal people, the low dose is 1 / 2 of the middle dose; the high dose is 2 times of the middle dose. The low, middle and high drug-containing serum contents are the mesenchymal stem cells prepared by giving different doses of the composite water extract according to the body weight of rats as described in Example 1: 0.315 mg / g, 0.63 mg / g, and 1.26 mg / g.

[0089] The model group and the normal group were given normal saline 1 mL / time / week for 4 consecutive weeks during lateral ventricular puncture; the cell intervention group was given cell preparations for lateral ventricular puncture, 1 mL / time / week for 4 consecutive weeks.

[0090] Water maze experiment: Morris water maze was used to train and evaluate the spatial learning and cognitive function of mice. The water maze is divided into 4 quadrants (NW, NE, SW, SE). A 5.0cm×5.0cm circular platform is placed in the SW quadrant, and the platform is placed 2cm below the liquid surface. A camera with a display system is placed above the water maze, placed in a quiet, continuously illuminated place, and the water temperature is maintained at 23-25°C. The water maze test was started one week after the last administration of the mice. First, 6d of water maze training was performed, 60s / time. If the mouse did not reach the platform during 60s, a guide rod was used to help the mouse reach the platform, and the mouse stayed for 20s after reaching the platform. After the training, the same training procedure was used to perform 6 cognitive function tests. The mouse's movement trajectory was tracked by a camera, and the SMART 3.0 software system recorded the movement time and distance. The results are shown in Table 2.

[0091] Table 2 Effects of different doses of Chinese medicine on the escape latency, number of crossing the original platform and the time spent in the original platform quadrant of rats

[0092]

[0093] Note: *Compared with the normal group, P<0.05; **Compared with the model group, P<0.01; #Compared with the model group, P<0.05; ##Compared with the model group, P<0.01;

[0094] As shown in Table 2, there are extremely significant differences between the normal group and the model group, P < 0.01, indicating that the model was successfully established. 6 cells, 5×10 6 cells, 1×10 7There was no significant difference in the data of the cells compared with the normal group, P>0.05. Compared with the data of the model group, there were extremely significant differences, P<0.01. This shows that low-dose drug-containing serum combined with various doses of stem cells can effectively improve the behavior of PSCI rats. There was no difference in learning and memory ability between the treated rats and the normal group rats. In the medium-dose group, the cell doses were 1×10 6 cells, 5×10 6 cells, 1×10 7 There was no significant difference in the data of the blank group (P>0.05). There was a very significant difference in the data of the model group (P<0.01). This showed that the medium-dose serum combined with various doses of stem cells could effectively improve the behavior of PSCI rats. There was no difference in learning and memory ability between the treated rats and the normal group rats. In the high-dose group, the cell doses were 1×10 6 cells, 5×10 6 cells, 1×10 7 There was no significant difference in the data of cells compared with the blank group, P>0.05. Compared with the data of the model group, there were extremely significant differences, P<0.01, indicating that high-dose medicated serum combined with various doses of stem cells can effectively improve the behavior of PSCI rats, and there is no difference in learning and memory ability between the treated rats and the normal group rats. In summary, umbilical cord mesenchymal stem cells cultured in hypoxia with medicated serum can have a significant therapeutic effect on PSCI disease.

[0095] Example 5 Screening of the best Chinese medicine formula for treating post-stroke cognitive impairment (PSCI)

[0096] Preparation of composite water extract (Chifukang decoction): The whole prescription components: 6g of ginseng, 30g of astragalus, 15g of polygonum multiflorum, 15g of angelica, 15g of Morinda officinalis, 6g of gastrodia, 6g of polygala, 6g of pinellia, and 6g of calamus were placed in a decoction pot, 1200mL of water was added, and after soaking in distilled water for 30min, it was boiled over high heat and then turned to low heat to continue decocting until 600mL remained, filtered while hot, and the filtrate was concentrated by rotary evaporation to a concentrated solution with a synthetic medicinal material of 132.3mg / mL to obtain a composite water extract, and the medicinal liquid was placed in a 50mL centrifuge tube and stored in a refrigerator at 4°C for standby use. The above composite water extract was commissioned to be produced and provided by Kangmei Pharmaceutical Co., Ltd. According to the method of Example 3, PSCI rats were modeled and treated by gavage according to the weight of rats using the Chinese medicine formula, and the dosage for rats was 13.23mg / g / day; the gavage frequency was 1 time / day for 7 consecutive days. The spatial learning and memory abilities of the rats were then tested using the Morris water maze test.

[0097] Comparative Example 1: Prescription without a sovereign drug component

[0098] Compared with Example 5, the Chinese medicine formula does not include ginseng ingredients, and only includes 30g of Astragalus, 15g of Polygonum multiflorum, 15g of Angelica sinensis, 15g of Morinda officinalis, 6g of Gastrodia elata, 6g of Polygala tenuifolia, 6g of Pinellia ternata, and 6g of Acorus calamus. The rest of the preparation method is the same as Example 5.

[0099] Comparative Example 2: Prescription without the ministerial drug component

[0100] Compared with Example 5, the Chinese medicine formula only includes 6g of ginseng, 6g of gastrodia elata, 6g of polygala tenuifolia, 6g of pinellia ternata, and 6g of calamus tatarinowii, and the rest of the preparation method is the same as Example 5.

[0101] Comparative Example 3: Prescription without main drug and auxiliary drug components

[0102] Compared with Example 5, the Chinese medicine formula only includes 6g of Gastrodia elata, 6g of Polygala tenuifolia, 6g of Pinellia ternata, and 6g of Acorus gramineus, and the rest of the preparation method is the same as Example 5.

[0103] The spatial learning and memory abilities of the rats treated with Example 5 and Comparative Examples 1-3 were tested by Morris water maze test, and the effects of different prescription compositions on the escape latency, the number of crossing the original platform, and the quadrant residence time of the PSCI rats were recorded.

[0104] The Morris water maze test was used to test the spatial learning and memory abilities of rats as follows:

[0105] A. Acquisition phase: Divide the water maze pool into four quadrants, and place the platform in the center of one of the quadrants. Place the rat in the water with its head facing the pool wall, and record the time (s) it takes for the animal to find the underwater platform. Continue training for 5 days.

[0106] B. Orienteering experiment: Rats were placed in water facing the pool wall in the order of quadrant III-quadrant I-quadrant IV-quadrant II. The time taken for the rats to climb onto the platform within 90 s and stay there for 3 s was recorded, which was the escape latency (s). For rats that took more than 90 s, the escape latency was recorded as 90 s.

[0107] C. Spatial exploration experiment After the directional navigation test was completed, the platform was immediately removed and the rat was placed in the water from the first quadrant opposite to the original platform, facing the pool wall. The number of times the rat crossed the original platform (times) and the time (s) the rat stayed in the original platform quadrant were recorded within 90 seconds.

[0108] Table 3 Effects of different prescription compositions on escape latency, number of crossing the original platform and quadrant residence time of the original platform in PSCI rats

[0109]

[0110] Note: *Compared with the normal group, P<0.05; **Compared with the model group, P<0.01; #Compared with the model group, P<0.05; ##Compared with the model group, P<0.01;

[0111] The effects of different prescription compositions on the escape latency, number of crossings of the original platform, and the time spent in the original platform quadrant of PSCI rats are shown in Table 3. It can be seen from Table 3 that the normal group and the model group have extremely significant differences, P<0.01, indicating that the modeling is successful. The whole group of prescriptions are compared with the normal group, with significant differences, P<0.05; the whole group of prescriptions are compared with the data of the model group, with significant differences, P<0.05. It shows that the whole group of prescriptions can improve the behavior of PSCI rats to a certain extent, but still fail to produce a good therapeutic effect. The comparison of the removal of the monarch drug component in Example 1 with the normal group has an extremely significant difference, P<0.01; the comparison of the removal of the monarch drug component in Example 1 with the data of the model group does not have a significant difference, P>0.05, indicating that the removal of the monarch drug component cannot improve the behavior of PSCI rats. Comparative Example 2, when the ministerial drug components were removed, was compared with the normal group, and there was a very significant difference, P<0.01; Comparative Example 2, when the ministerial drug components were removed, was compared with the data of the model group, and there was no significant difference, P>0.05. This shows that the removal of the ministerial drug components in Comparative Example 2 cannot improve the behavior of PSCI rats. Comparative Example 3, when the monarch and ministerial drug components were removed, was compared with the normal group, and there was a very significant difference, P<0.01; Comparative Example 3, when the monarch and ministerial drug components were removed, was compared with the data of the model group, there was no significant difference, P>0.05. This shows that the removal of the monarch and ministerial drug components in Comparative Example 3 cannot improve the behavior of PSCI rats. In summary, 6g of ginseng, 30g of astragalus, 15g of polygonum multiflorum, 15g of angelica, 15g of Morinda officinalis, 6g of Gastrodia elata, 6g of Polygala tenuifolia, 6g of French pinellia, and 6g of Acorus gramineus are the best prescriptions for the treatment of post-stroke cognitive impairment.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A drug-containing serum culture medium, characterized in that The drug-containing serum culture medium comprises a conditioned medium and drug-containing serum; the drug-containing serum is serum obtained by intragastric administration of a Chinese medicine formula extract to treat rats; the Chinese medicine formula comprises the following components by weight: 20-40 parts of Astragalus, 5-7 parts of Ginseng, 10-20 parts of Polygonum multiflorum, 10-20 parts of Angelica sinensis, 5-7 parts of Gastrodia elata, 5-7 parts of Polygala tenuifolia, 10-20 parts of Morinda officinalis, 5-7 parts of Pinellia ternata, and 5-7 parts of Acorus calamus; The method for preparing the conditioned medium is as follows: after culturing umbilical vein endothelial cell P3 cells with complete medium for 24-56 hours, separating the culture system supernatant to obtain the conditioned medium; The preparation of the Chinese medicine formula extract comprises the following steps: weighing the Chinese medicine formula, adding 1000-1200 mL of water, soaking for 25-30 minutes and then boiling over high heat, then decocting over low heat until 500-600 mL is reached and filtering while hot, and concentrating the filtrate to obtain the Chinese medicine formula extract.

2. The culture medium according to claim 1, characterized in that The preparation of the drug-containing serum comprises the following steps: Rats were treated with the extract of the Chinese medicine formula by gavage, and blood was collected from the rats after the treatment, and drug-containing serum was obtained after purification; the frequency of the gavage treatment was 3 times a day for 7 consecutive days; in the gavage treatment, the dosage of the extract of the Chinese medicine formula was 5 mL / time.

3. The culture medium according to claim 1, characterized in that The volume ratio of the conditioned medium to the drug-containing serum is conditioned medium: drug-containing serum = 1: (0.2-0.3).

4. A method for preparing umbilical cord mesenchymal stem cells, characterized in that: The following steps are involved: (1) Primary culture and subculture of umbilical cord mesenchymal stem cells: Isolate the Wharton's jelly in the umbilical cord, separate the umbilical cord mesenchymal stem cells after primary culture, and continue subculture to obtain P3-P5 umbilical cord mesenchymal stem cells; (2) Hypoxia treatment with medicated serum: Take the P3-P5 umbilical cord mesenchymal stem cells described in step (1), culture them under hypoxic conditions using the medicated serum culture medium described in any one of claims 1-3 until the cell fusion rate reaches 90%, collect and resuspend the cells, and obtain umbilical cord mesenchymal stem cells with enhanced differentiation ability.

5. The method according to claim 4, characterized in that In step (2), the hypoxic condition is that the cells are cultured at an oxygen content of 1%-3%.

6. Use of the method for preparing umbilical cord mesenchymal stem cells according to claim 4 or 5 in preparing a drug for treating post-stroke cognitive impairment.

7. The use according to claim 6, characterized in that The concentration of umbilical cord mesenchymal stem cells in the drug for treating post-stroke cognitive impairment is 1×10 6 cells / mL-1×10 7 cells / mL.

8. The use according to claim 6, characterized in that The drug for treating post-stroke cognitive impairment is administered by injection; the frequency of the injection is 0.1-1.0 mL / time / week, and the administration is continued for 3-5 weeks; the injection site is the lateral ventricle of the brain.

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