Stem cell preparation for treating cerebral apoplexy and preparation method thereof

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses a stem cell preparation for treating cerebral apoplexy and a preparation method thereof. The stem cell preparation for treating the cerebral apoplexy is prepared by combining optimized umbilical cord mesenchymal stem cells and traditional Chinese medicine microcapsules, and the umbilical cord mesenchymal stem cells are optimized by using a functional aid formed by compounding panax notoginseng saponins, alkannin and catechin. Natural traditional Chinese medicines of gastrodia elata, radix puerariae and saussurea involucrata are subjected to multiple extraction with absolute ethyl alcohol and a bacterium enzyme solution to prepare the traditional Chinese medicine microcapsule. The natural active ingredients and the stem cells play a synergistic role, and the prepared stem cell preparation has the advantages of high stability and high safety, has a remarkable targeted treatment effect on ischemic cerebral apoplexy, and has an important application value in prevention and treatment of cardiovascular and cerebrovascular diseases.

Description

Technical Field

[0001] The present application relates to the field of biomedical technology, and more specifically, to a stem cell preparation for treating cerebral stroke and a preparation method thereof. Background Art

[0002] Stroke, commonly known as stroke, is divided into two types: ischemic stroke and hemorrhagic stroke. It is a disease caused by various reasons that damage cerebral blood vessels and produce focal or overall brain tissue damage. The typical symptoms of ischemic stroke are weakness or numbness of one limb, numbness of one side of the face or crooked corner of the mouth, slurred speech or difficulty understanding language, loss or blurring of vision in one or both eyes, impaired consciousness or convulsions, etc. The typical symptoms of hemorrhagic stroke are headache, nausea, vomiting, varying degrees of impaired consciousness and limb paralysis. At present, the treatment of cerebral stroke usually adopts the methods of drug therapy and surgical treatment. However, drug treatment is unstable and the human body is prone to adverse reactions, and surgical treatment will also leave sequelae.

[0003] Stem cells are a type of cell that has unlimited or immortal self-renewal capabilities and can produce at least one type of highly differentiated daughter cells. As an emerging treatment method, stem cell therapy has extremely broad application prospects in the fields of cell repair, developmental biology, and pharmacology in life sciences. Stroke has the characteristics of high incidence, disability, recurrence, and mortality. It is the leading cause of death among Chinese residents, and the incidence of stroke in China is increasing year by year. Therefore, it is urgently necessary to provide a stem cell preparation for the treatment of stroke and its preparation method. Summary of the invention

[0004] In order to solve the problems of instability, adverse reactions and postoperative sequelae in traditional drug and surgical treatments, the present application provides a stem cell preparation for the treatment of cerebral stroke and a preparation method thereof.

[0005] In a first aspect, the present application provides a method for preparing a stem cell preparation for treating cerebral stroke, comprising the following steps: S1. Inoculate the optimized umbilical cord mesenchymal stem cells in serum-free medium and then culture them in an incubator. When the cell confluence reaches 70%-90%, digest and passage them with trypsin; S2, digesting the optimized umbilical cord mesenchymal stem cells passaged to the fourth generation with trypsin, adding DMEM medium containing fetal bovine serum to terminate the digestion, centrifuging the medium, adding new DMEM medium containing fetal bovine serum to resuspend the cells to prepare a cell suspension, and then diluting the cell suspension with physiological saline; S3. Add Chinese medicine microcapsules and sodium alginate to the diluted cell suspension, and centrifuge to obtain a stem cell preparation.

[0006] Preferably, the incubator in step S1 is a 37° C., 5% CO 2 incubator.

[0007] Preferably, in step S1, the optimized umbilical cord mesenchymal stem cells are prepared by the following method: (1) dissolving the functional additive in an ethanol solution and adding the solution to a serum-free culture medium to prepare a culture medium containing the functional additive; (2) Inoculate the umbilical cord mesenchymal stem cells into a culture vessel, and after the cells are completely attached to the wall, add a culture medium containing functional additives, and culture the cells for 2-4 days to obtain optimized umbilical cord mesenchymal stem cells.

[0008] Preferably, the mass concentration of the ethanol solution in step (1) is 10%-30%.

[0009] Preferably, in step (1), the mass ratio of the functional auxiliary agent to the ethanol solution is 1-3:10.

[0010] Preferably, the content of the functional additive in the culture medium containing the functional additive in step (1) is 20-100 μM.

[0011] Preferably, the functional auxiliary agent in step (1) is composed of notoginseng total saponins, shikonin and catechins mixed in a mass ratio of 3-5:1:1.2-1.6.

[0012] Preferably, in step S3, the Chinese medicine microcapsules are prepared by the following method: 1) Gastrodia elata, Pueraria root and snow lotus are mixed, ground into powder, sieved, stirred and mixed with anhydrous ethanol, and ultrasonically dispersed to obtain the primary extract; 2) Add bacterial enzyme solution to the primary extract, stir and mix, seal and ferment, and centrifuge to obtain the Chinese medicine extract; 3) The Chinese medicine extract and chitosan are stirred and mixed, ultrasonically dispersed, and then homogenized to form an emulsion, and calcium chloride solution is added, stirred and mixed, and then freeze-dried to obtain Chinese medicine microcapsules.

[0013] Preferably, in step 1), the mass ratio of Gastrodia elata, Pueraria lobata, Saussurea involucrata and anhydrous ethanol is 10-14:5-7:1-3:50.

[0014] Preferably, in step 2), the mass ratio of the primary extract to the bacterial enzyme solution is 1:0.3-0.7.

[0015] Preferably, the bacterial enzyme solution in step 2) is prepared by the following method: according to the mass ratio of composite microorganisms, papain and water being 10-12:1-3:30, the composite microorganisms, papain and water are stirred and mixed to obtain the bacterial enzyme solution.

[0016] Preferably, the composite microorganism is composed of a mixture of Lactobacillus acidophilus and Bacillus licheniformis with a live cell count ratio of 1-1.4:1.5-1.9.

[0017] Preferably, in step 3), the mass ratio of the Chinese medicine extract, chitosan and calcium chloride solution is 1:1-3:0.5-0.9.

[0018] Preferably, the mass concentration of the calcium chloride solution in step 3) is 10%-30%.

[0019] Preferably, in step S3, the mass ratio of the diluted cell suspension, the traditional Chinese medicine microcapsules and the sodium alginate is 1-3:1:0.1-0.3.

[0020] In a second aspect, the present application provides a stem cell preparation for treating cerebral stroke, which is prepared by the above method.

[0021] In summary, this application has the following beneficial effects: 1. This application uses a variety of natural ingredients to prepare Chinese medicine microcapsules through reasonable raw material matching, raw material ratio and process parameters, and uses Chinese medicine microcapsules and optimized umbilical cord mesenchymal stem cells to prepare stem cell preparations, so that the natural active ingredients and stem cells can play a synergistic role, improve the stability and safety of cerebral stroke treatment, and have a significant targeted therapeutic effect on ischemic cerebral stroke.

[0022] 2. The present application adds functional adjuvants composed of notoginseng total saponins, shikonin and catechins to the culture medium of umbilical cord mesenchymal stem cells. Notoginseng total saponins can invigorate blood circulation, remove blood stasis, dredge meridians and activate collaterals, effectively improve cardiovascular and cerebrovascular functions, and promote the proliferation and differentiation of stem cells; shikonin and catechins can scavenge free radicals and play anti-inflammatory and antiviral effects. In addition, shikonin can increase the fat solubility of catechins, thereby increasing the stability and bioavailability of catechins, thereby providing cells with a more stable and healthy growth environment, promoting the differentiation and proliferation of stem cells, and increasing the activity of stem cells. The combination of the three can produce a synergistic effect and enhance pharmacological activity.

[0023] 3. This application uses Gastrodia elata, Pueraria root and Snow Lotus as co-medicines. The active ingredients such as gastrodin and gastrodia polysaccharide in Gastrodia elata and the active ingredients such as isoflavones and pueraria glycosides in Pueraria root can improve cerebral blood circulation, relieve cerebral vasospasm, increase blood flow, have a certain anti-thrombotic effect, and can promote brain tissue recovery; the active ingredients such as alkaloids and flavonoids in Snow Lotus can dispel wind and dampness, promote blood circulation and dredge collaterals, and can relieve symptoms such as joint pain and limb numbness caused by stroke. The three compounds can complement each other and synergistically enhance the overall pharmacological effect.

[0024] 4. When preparing Chinese medicine microcapsules, the present application first uses an ethanol solution to perform preliminary extraction on the Chinese medicine raw materials, and then adds a bacterial enzyme solution for fermentation extraction. Multiple extractions can improve the purity of the active ingredients. The bacterial enzyme solution is prepared by mixing composite microorganisms and papain. The addition of composite microorganisms can degrade certain macromolecular substances in Chinese medicine into small molecular substances, promote the release of active ingredients of Chinese medicine, enhance the interaction between active ingredients, and improve the efficacy; on the other hand, it can also increase the content of active ingredients in Chinese medicine, produce a variety of metabolites that are beneficial to the human body, and add new efficacy to Chinese medicine. The synergistic effect of papain and composite microorganisms can not only significantly accelerate the fermentation process and improve production efficiency, but also improve the absorption and utilization rate of active ingredients, while modifying active ingredients, producing new functional substances, and improving the nutritional value and biological activity of fermentation products.

[0025] 5. The present application prepares Chinese medicine into microcapsules to achieve the homogenization of Chinese medicine ingredients, so that various ingredients can be uniformly absorbed by the human body. At the same time, the uniformity of Chinese medicine ingredients enables stem cells to obtain a stable and consistent pharmacological environment, which is beneficial to the growth and function of stem cells; secondly, Chinese medicine microcapsule technology can retain the biologically active substances and nutrients of Chinese medicine to the maximum extent, and can also significantly improve the bioavailability of drugs, making the dispersion and dissolution of drugs in the body more rapid and sufficient, thereby improving the efficiency of drug absorption by the human body. DETAILED DESCRIPTION

[0026] The present application is further described in detail below with reference to the embodiments.

[0027] Embodiment 1-5 provides a stem cell preparation for treating cerebral stroke and a preparation method thereof.

[0028] Umbilical cord mesenchymal stem cells (purchased from Hebei Beike Biotechnology Co., Ltd.), Panax notoginseng saponins (CAS No.: 88105-29-7, purchased from Nanjing Jiadi Biotechnology Co., Ltd.), shikonin (CAS No.: 517-89-5, purchased from Nanjing Qiushi Pharmaceutical Technology Co., Ltd.), catechins (CAS No.: 18829-70-4, purchased from Shandong Siyang Biotechnology Co., Ltd.), serum-free culture medium (registration number: 038476, purchased from Beijing Huakan Biotechnology Co., Ltd.), DMEM culture medium (catalog number: BC-M-030, purchased from Nanjing Shenghang Biotechnology Co., Ltd.), pancreatin (model: PH0502, purchased from Shanghai Pu Yu Industrial Technology Co., Ltd.), Lactobacillus acidophilus is Lactobacillus acidophilus BWCC61818, Bacillus licheniformis is Bacillus licheniformis ACCC10236 (all purchased from Henan Wanjia Standard Material Research and Development Center), papain (CAS No.: 9001-73-4, purchased from Hefei Hongrui Biotechnology Co., Ltd.), sodium alginate (CAS No.: 9005-38-3, purchased from Shenzhen Love Biotechnology Co., Ltd.).

[0029] Example 1 A method for preparing a stem cell preparation for treating cerebral stroke comprises the following steps: S1. Inoculate the optimized umbilical cord mesenchymal stem cells in serum-free medium and then culture them in a 37°C, 5% CO2 incubator. When the cell confluence reaches 70%, digest and passage them with trypsin. S2. The optimized umbilical cord mesenchymal stem cells passaged to the fourth generation were digested with trypsin for 2 minutes, and then the digestion was terminated by adding DMEM medium containing 10% fetal bovine serum. The culture medium was centrifuged at 600r / min for 3 minutes, and then new DMEM medium containing 10% fetal bovine serum was added to resuspend the cells to prepare a cell suspension, and then physiological saline was added to dilute the cell suspension to a cell concentration of 10 5 Pieces / ml; S3. According to the mass ratio of the diluted cell suspension, Chinese medicine microcapsules and sodium alginate of 1:1:0.1, Chinese medicine microcapsules and sodium alginate were added to the diluted cell suspension, and the mixture was centrifuged at 800 r / min for 3 min to obtain a stem cell preparation.

[0030] The optimized umbilical cord mesenchymal stem cells are prepared by the following method: (1) The functional additive (composed of a mixture of notoginseng total saponins, shikonin and catechins in a mass ratio of 3:1:1.2) to the ethanol solution was 1:10, the functional additive and the ethanol solution with a mass concentration of 20% were stirred at 400 r / min for 10 min, and then added to the serum-free culture medium to prepare a culture medium containing the functional additive (the content of the functional additive was 20 μM); (2) The umbilical cord mesenchymal stem cells are inoculated into a culture vessel, and after the cells are completely attached to the wall, a culture medium containing a functional additive is added, and the cells are cultured for 2 days to obtain optimized umbilical cord mesenchymal stem cells.

[0031] The Chinese medicine microcapsule is prepared by the following method: 1) Gastrodia elata, Pueraria root, Snow lotus and anhydrous ethanol were mixed in a mass ratio of 10:5:1:50, ground into powder, passed through a 100-mesh sieve, stirred with anhydrous ethanol at 600 r / min for 10 min, and then ultrasonically dispersed for 30 min under the control of ultrasonic frequency of 25 kHz and power of 500 W to obtain a primary extract; 2) Add the bacterial enzyme solution to the primary extract at a mass ratio of 1:0.3, stir at 500 r / min for 30 min, seal and ferment for 8 h, and then centrifuge at 10000 r / min for 30 min to obtain the Chinese medicine extract; 3) According to the mass ratio of Chinese medicine extract, chitosan and calcium chloride solution of 1:1:0.5, the Chinese medicine extract and chitosan were stirred at 400 r / min for 30 min, ultrasonically dispersed at 50°C for 10 min, the ultrasonic power was controlled to 500 W, and the ultrasonic frequency was 20 kHz, then homogenized 3 times at 10 MPa for 10 min each time to form an emulsion, and a calcium chloride solution with a mass concentration of 20% was added, stirred at 400 r / min for 10 min, and then freeze-dried at -10°C for 10 h to obtain Chinese medicine microcapsules.

[0032] The bacterial enzyme solution in step 2) is prepared by the following method: according to the mass ratio of composite microorganisms, papain and water being 10:1:30, composite microorganisms (composed of a mixture of Lactobacillus acidophilus and Bacillus licheniformis with a live cell count ratio of 1:1.5), papain and water are stirred and mixed at a speed of 600 r / min for 30 minutes to obtain the bacterial enzyme solution.

[0033] Example 2 A method for preparing a stem cell preparation for treating cerebral stroke comprises the following steps: S1. Inoculate the optimized umbilical cord mesenchymal stem cells in serum-free medium and then culture them in a 37°C, 5% CO2 incubator. When the cell confluence reaches 75%, digest and passage them with trypsin. S2. The optimized umbilical cord mesenchymal stem cells passaged to the fourth generation were digested with trypsin for 2 minutes, and then the digestion was terminated by adding DMEM medium containing 10% fetal bovine serum. The culture medium was centrifuged at 650r / min for 3.5 minutes, and then new DMEM medium containing 10% fetal bovine serum was added to resuspend the cells into a cell suspension, and then physiological saline was added to dilute the cell suspension to a cell concentration of 10 5 Pieces / ml; S3. According to the mass ratio of the diluted cell suspension, Chinese medicine microcapsules and sodium alginate of 1.5:1:0.15, Chinese medicine microcapsules and sodium alginate were added to the diluted cell suspension, and the mixture was centrifuged at 850 r / min for 3.5 min to obtain a stem cell preparation.

[0034] The optimized umbilical cord mesenchymal stem cells are prepared by the following method: (1) The mass ratio of the functional additive (composed of a mixture of Panax notoginseng total saponins, shikonin and catechins in a mass ratio of 3.5:1:1.3) to the ethanol solution was 1.5:10, the functional additive and the ethanol solution with a mass concentration of 20% were stirred at 450 r / min for 15 min, and then added to the serum-free culture medium to prepare a culture medium containing the functional additive (the content of the functional additive was 20 μM); (2) The umbilical cord mesenchymal stem cells are inoculated into a culture vessel, and after the cells are completely attached to the wall, a culture medium containing a functional additive is added, and the cells are cultured for 2.5 days to obtain optimized umbilical cord mesenchymal stem cells.

[0035] The Chinese medicine microcapsule is prepared by the following method: 1) Gastrodia elata, Pueraria root, Snow lotus and anhydrous ethanol were mixed in a mass ratio of 11:5.5:1.5:50, ground into powder, passed through a 105-mesh sieve, stirred with anhydrous ethanol at 650 r / min for 15 min, and then ultrasonically dispersed for 35 min at a frequency of 25 kHz and a power of 500 W to obtain a primary extract; 2) Add the bacterial enzyme solution to the primary extract at a mass ratio of 1:0.4, stir at 500 r / min for 30 min, seal and ferment for 8.5 h, and then centrifuge at 10500 r / min for 30 min to obtain the Chinese medicine extract; 3) According to the mass ratio of Chinese medicine extract, chitosan and calcium chloride solution of 1:1.5:0.6, the Chinese medicine extract and chitosan were stirred at 450 r / min for 35 min, ultrasonically dispersed at 50°C for 15 min, the ultrasonic power was controlled to 500 W, and the ultrasonic frequency was 20 kHz, then homogenized 3 times at 15 MPa for 10 min each time to form an emulsion, and a calcium chloride solution with a mass concentration of 20% was added, stirred at 400 r / min for 10 min, and then freeze-dried at -10°C for 11 h to obtain Chinese medicine microcapsules.

[0036] Wherein, the bacterial enzyme liquid in step 2) is prepared by the following method: according to the mass ratio of composite microorganisms, papain and water being 10.5:1.5:30, the composite microorganisms (composed of a mixture of Lactobacillus acidophilus and Bacillus licheniformis with a live cell count ratio of 1.1:1.6), papain and water are stirred and mixed at a speed of 600 r / min for 35 minutes to obtain the bacterial enzyme liquid.

[0037] Example 3 A method for preparing a stem cell preparation for treating cerebral stroke comprises the following steps: S1. Inoculate the optimized umbilical cord mesenchymal stem cells in serum-free medium and then culture them in a 37°C, 5% CO2 incubator. When the cell confluence reaches 80%, digest and passage them with trypsin. S2. The optimized umbilical cord mesenchymal stem cells passaged to the fourth generation were digested with trypsin for 2 minutes, and then the digestion was terminated by adding DMEM medium containing 10% fetal bovine serum. The culture medium was centrifuged at 700r / min for 4 minutes, and then new DMEM medium containing 10% fetal bovine serum was added to resuspend the cells to prepare a cell suspension, and then physiological saline was added to dilute the cell suspension to a cell concentration of 10 5 Pieces / ml; S3. According to the mass ratio of the diluted cell suspension, Chinese medicine microcapsules and sodium alginate of 2:1:0.2, Chinese medicine microcapsules and sodium alginate were added to the diluted cell suspension, and the mixture was centrifuged at 900 r / min for 4 min to obtain a stem cell preparation.

[0038] The optimized umbilical cord mesenchymal stem cells are prepared by the following method: (1) The functional additive (composed of a mixture of notoginseng total saponins, shikonin and catechins in a mass ratio of 4:1:1.4) to the ethanol solution was 2:10, the functional additive and the ethanol solution with a mass concentration of 20% were stirred at a speed of 500 r / min for 20 min, and then added to the serum-free culture medium to prepare a culture medium containing the functional additive (the content of the functional additive was 20 μM); (2) The umbilical cord mesenchymal stem cells are inoculated into a culture vessel, and after the cells are completely attached to the wall, a culture medium containing functional additives is added, and the cells are cultured for 3 days to obtain optimized umbilical cord mesenchymal stem cells.

[0039] The Chinese medicine microcapsule is prepared by the following method: 1) Gastrodia elata, Pueraria root, Snow lotus and anhydrous ethanol were mixed in a mass ratio of 12:6:2:50, ground into powder, passed through a 110-mesh sieve, stirred with anhydrous ethanol at 700 r / min for 20 min, and then ultrasonically dispersed for 40 min at a frequency of 25 kHz and a power of 500 W to obtain a primary extract; 2) Add 0.5 parts of the bacterial enzyme solution to the primary extract at a mass ratio of 1:0.5, stir at 500 r / min for 30 min, seal and ferment for 9 h, and then centrifuge at 11000 r / min for 30 min to obtain the Chinese medicine extract; 3) According to the mass ratio of Chinese medicine extract, chitosan and calcium chloride solution of 1:2:0.7, the Chinese medicine extract and chitosan were stirred at 500 r / min for 40 min, ultrasonically dispersed at 50°C for 20 min, the ultrasonic power was controlled to 500 W, and the ultrasonic frequency was 20 kHz, then homogenized 3 times at 20 MPa for 10 min each time to form an emulsion, and a calcium chloride solution with a mass concentration of 20% was added, stirred at 400 r / min for 10 min, and then freeze-dried at -10°C for 12 h to obtain Chinese medicine microcapsules.

[0040] Wherein, the bacterial enzyme solution in step 2) is prepared by the following method: according to the mass ratio of composite microorganisms, papain and water being 11:2:30, composite microorganisms (composed of a mixture of Lactobacillus acidophilus and Bacillus licheniformis with a live cell count ratio of 1.2:1.7), papain and water are stirred and mixed at a speed of 600 r / min for 40 minutes to obtain the bacterial enzyme solution.

[0041] Example 4 A method for preparing a stem cell preparation for treating cerebral stroke comprises the following steps: S1. Inoculate the optimized umbilical cord mesenchymal stem cells in serum-free medium and then culture them in a 37°C, 5% CO2 incubator. When the cell confluence reaches 85%, digest and passage them with trypsin. S2. The optimized umbilical cord mesenchymal stem cells passaged to the fourth generation were digested with trypsin for 2 minutes, and then the digestion was terminated by adding DMEM medium containing 10% fetal bovine serum. The culture medium was centrifuged at 750 r / min for 4.5 minutes, and then new DMEM medium containing 10% fetal bovine serum was added to resuspend the cells to prepare a cell suspension, and then physiological saline was added to dilute the cell suspension to a cell concentration of 10 5 Pieces / ml; S3. According to the mass ratio of the diluted cell suspension, Chinese medicine microcapsules and sodium alginate of 2.5:1:0.25, Chinese medicine microcapsules and sodium alginate were added to the diluted cell suspension, and the mixture was centrifuged at 950 r / min for 4.5 min to obtain a stem cell preparation.

[0042] The optimized umbilical cord mesenchymal stem cells are prepared by the following method: (1) The mass ratio of the functional additive (composed of a mixture of Panax notoginseng total saponins, shikonin and catechins in a mass ratio of 4.5:1:1.5) to the ethanol solution was 2.5:10, the functional additive and the ethanol solution with a mass concentration of 20% were stirred at a speed of 550 r / min for 25 min, and then added to the serum-free culture medium to prepare a culture medium containing the functional additive (the content of the functional additive was 20 μM); (2) The umbilical cord mesenchymal stem cells are inoculated into a culture vessel, and after the cells are completely attached to the wall, a culture medium containing functional additives is added, and the cells are cultured for 3.5 days to obtain optimized umbilical cord mesenchymal stem cells.

[0043] The Chinese medicine microcapsule is prepared by the following method: 1) Gastrodia elata, Pueraria root, Snow lotus and anhydrous ethanol were mixed in a mass ratio of 13:6.5:2.5:50, ground into powder, passed through a 115-mesh sieve, stirred with anhydrous ethanol at 750 r / min for 25 min, and then ultrasonically dispersed for 45 min at a frequency of 25 kHz and a power of 500 W to obtain a primary extract; 2) Add the bacterial enzyme solution to the primary extract at a mass ratio of 1:0.6, stir at 500 r / min for 30 min, seal and ferment for 9.5 h, and then centrifuge at 11500 r / min for 30 min to obtain the Chinese medicine extract; 3) According to the mass ratio of Chinese medicine extract, chitosan and calcium chloride solution of 1:2.5:0.8, the Chinese medicine extract and chitosan were stirred at 550 r / min for 45 min, ultrasonically dispersed at 50°C for 25 min, the ultrasonic power was controlled to 500 W, and the ultrasonic frequency was 20 kHz, then homogenized 3 times at 25 MPa for 10 min each time to form an emulsion, and a calcium chloride solution with a mass concentration of 20% was added, stirred at 400 r / min for 10 min, and then freeze-dried at -10°C for 13 h to obtain Chinese medicine microcapsules.

[0044] The bacterial enzyme solution in step 2) is prepared by the following method: according to the mass ratio of composite microorganisms, papain and water being 11.5:2.5:30, composite microorganisms (composed of a mixture of Lactobacillus acidophilus and Bacillus licheniformis with a live cell count ratio of 1.3:1.8), papain and water are stirred and mixed at a speed of 600 r / min for 45 minutes to obtain the bacterial enzyme solution.

[0045] Example 5 A method for preparing a stem cell preparation for treating cerebral stroke comprises the following steps: S1. Inoculate the optimized umbilical cord mesenchymal stem cells in serum-free medium and then culture them in a 37°C, 5% CO2 incubator. When the cell confluence reaches 90%, digest and passage them with trypsin. S2. The optimized umbilical cord mesenchymal stem cells passaged to the fourth generation were digested with trypsin for 2 minutes, and then the digestion was terminated by adding DMEM medium containing 10% fetal bovine serum. The culture medium was centrifuged at 800r / min for 5 minutes, and then new DMEM medium containing 10% fetal bovine serum was added to resuspend the cells into a cell suspension, and then physiological saline was added to dilute the cell suspension to a cell concentration of 10 5 Pieces / ml; S3. According to the mass ratio of the diluted cell suspension, Chinese medicine microcapsules and sodium alginate being 3:1:0.3, Chinese medicine microcapsules and sodium alginate were added to the diluted cell suspension, and the mixture was centrifuged at 1000 r / min for 5 min to obtain a stem cell preparation.

[0046] The optimized umbilical cord mesenchymal stem cells are prepared by the following method: (1) The functional additive (composed of a mixture of notoginseng total saponins, shikonin and catechins in a mass ratio of 5:1:1.6) to the ethanol solution was 3:10, the functional additive and the ethanol solution with a mass concentration of 20% were stirred at 600 r / min for 30 min, and then added to the serum-free culture medium to prepare a culture medium containing the functional additive (the content of the functional additive was 20 μM); (2) The umbilical cord mesenchymal stem cells are inoculated into a culture vessel, and after the cells are completely attached to the wall, a culture medium containing a functional additive is added, and the cells are cultured for 4 days to obtain optimized umbilical cord mesenchymal stem cells.

[0047] The Chinese medicine microcapsule is prepared by the following method: 1) Gastrodia elata, Pueraria root, Snow lotus and anhydrous ethanol were mixed in a mass ratio of 14:7:3:50, ground into powder, passed through a 120-mesh sieve, stirred with anhydrous ethanol at 800 r / min for 30 min, and then ultrasonically dispersed for 50 min at a frequency of 25 kHz and a power of 500 W to obtain a primary extract; 2) Add the bacterial enzyme solution to the primary extract at a mass ratio of 1:0.7, stir at 500 r / min for 30 min, seal and ferment for 10 h, and then centrifuge at 12000 r / min for 30 min to obtain the Chinese medicine extract; 3) According to the mass ratio of Chinese medicine extract, chitosan and calcium chloride solution of 1:3:0.9, the Chinese medicine extract and chitosan were stirred at 600 r / min for 50 min, ultrasonically dispersed at 50°C for 30 min, the ultrasonic power was controlled to 500 W, and the ultrasonic frequency was 20 kHz, then homogenized 3 times at 30 MPa for 10 min each time to form an emulsion, and a calcium chloride solution with a mass concentration of 20% was added, stirred at 400 r / min for 10 min, and then freeze-dried at -10°C for 14 h to obtain Chinese medicine microcapsules.

[0048] The bacterial enzyme solution in step 2) is prepared by the following method: according to the mass ratio of composite microorganisms, papain and water being 12:3:30, composite microorganisms (composed of a mixture of Lactobacillus acidophilus and Bacillus licheniformis with a live cell count ratio of 1.4:1.9), papain and water are stirred and mixed at a speed of 600 r / min for 50 minutes to obtain the bacterial enzyme solution.

[0049] Comparative Example 1 Comparative Example 1 is the same as Example 1, except that the functional additive does not contain shikonin, as follows: The optimized umbilical cord mesenchymal stem cells are prepared by the following method: (1) The functional additive (composed of a mixture of notoginseng total saponins and catechins in a mass ratio of 3:2.2) to the ethanol solution was 1:10, the functional additive and the ethanol solution with a mass concentration of 20% were stirred at 400 r / min for 10 min, and then added to the serum-free culture medium to prepare a culture medium containing the functional additive (the content of the functional additive was 20 μM); (2) The umbilical cord mesenchymal stem cells are inoculated into a culture vessel, and after the cells are completely attached to the wall, a culture medium containing a functional additive is added, and the cells are cultured for 2 days to obtain optimized umbilical cord mesenchymal stem cells.

[0050] Comparative Example 2 Comparative Example 2 is the same as Example 1, except that the functional additive does not contain catechins, as follows: The optimized umbilical cord mesenchymal stem cells are prepared by the following method: (1) The functional additive (composed of a mixture of Panax notoginseng total saponins and shikonin in a mass ratio of 3:2.2) to the ethanol solution was 1:10, the functional additive and the ethanol solution with a mass concentration of 20% were stirred at 400 r / min for 10 min, and then added to the serum-free culture medium to prepare a culture medium containing the functional additive (the content of the functional additive was 20 μM); (2) The umbilical cord mesenchymal stem cells are inoculated into a culture vessel, and after the cells are completely attached to the wall, a culture medium containing a functional additive is added, and the cells are cultured for 2 days to obtain optimized umbilical cord mesenchymal stem cells.

[0051] Comparative Example 3 Comparative Example 3 is the same as Example 1, except that the functional additive does not contain notoginseng total saponins, as follows: The optimized umbilical cord mesenchymal stem cells are prepared by the following method: (1) The functional auxiliary agent (composed of shikonin and catechin mixed in a mass ratio of 1:1.2) to the ethanol solution was 1:10, the functional auxiliary agent and the ethanol solution with a mass concentration of 20% were stirred at 400 r / min for 10 min, and then added to the serum-free culture medium to prepare a culture medium containing the functional auxiliary agent (the content of the functional auxiliary agent was 20 μM); (2) The umbilical cord mesenchymal stem cells are inoculated into a culture vessel, and after the cells are completely attached to the wall, a culture medium containing a functional additive is added, and the cells are cultured for 2 days to obtain optimized umbilical cord mesenchymal stem cells.

[0052] Comparative Example 4 Comparative Example 4 is the same as Example 1, except that the raw material of the Chinese medicine microcapsule does not contain Pueraria root, as follows: The Chinese medicine microcapsule is prepared by the following method: 1) Grind Gastrodia elata and Snow Lotus flower into powder according to the mass ratio of Gastrodia elata, Snow Lotus flower and anhydrous ethanol of 10:1:50, pass through a 100-mesh sieve, stir with anhydrous ethanol at 600 r / min for 10 min, and then control the ultrasonic frequency to 25 KHz and the power to 500 W for 30 min to obtain the primary extract; 2) Add the bacterial enzyme solution to the primary extract at a mass ratio of 1:0.3, stir at 500 r / min for 30 min, seal and ferment for 8 h, and then centrifuge at 10000 r / min for 30 min to obtain the Chinese medicine extract; 3) According to the mass ratio of Chinese medicine extract, chitosan and calcium chloride solution of 1:1:0.5, the Chinese medicine extract and chitosan were stirred at 400 r / min for 30 min, ultrasonically dispersed at 50°C for 10 min, the ultrasonic power was controlled to 500 W, and the ultrasonic frequency was 20 kHz, then homogenized 3 times at 10 MPa for 10 min each time to form an emulsion, and a calcium chloride solution with a mass concentration of 20% was added, stirred at 400 r / min for 10 min, and then freeze-dried at -10°C for 10 h to obtain Chinese medicine microcapsules.

[0053] The bacterial enzyme solution in step 2) is prepared by the following method: according to the mass ratio of composite microorganisms, papain and water being 10:1:30, composite microorganisms (composed of a mixture of Lactobacillus acidophilus and Bacillus licheniformis with a live cell count ratio of 1:1.5), papain and water are stirred and mixed at a speed of 600 r / min for 30 minutes to obtain the bacterial enzyme solution.

[0054] Comparative Example 5 Comparative Example 5 is the same as Example 1, except that the raw material of the Chinese medicine microcapsule does not contain snow lotus, as follows: The Chinese medicine microcapsule is prepared by the following method: 1) Gastrodia elata and Pueraria root were mixed in a mass ratio of 10:5:50, ground into powder, passed through a 100-mesh sieve, and stirred with anhydrous ethanol at 600 r / min for 10 min, and then ultrasonically dispersed for 30 min under a control of an ultrasonic frequency of 25 kHz and a power of 500 W to obtain a primary extract; 2) Add the bacterial enzyme solution to the primary extract at a mass ratio of 1:0.3, stir at 500 r / min for 30 min, seal and ferment for 8 h, and then centrifuge at 10000 r / min for 30 min to obtain the Chinese medicine extract; 3) According to the mass ratio of Chinese medicine extract, chitosan and calcium chloride solution of 1:1:0.5, the Chinese medicine extract and chitosan were stirred at 400 r / min for 30 min, ultrasonically dispersed at 50°C for 10 min, the ultrasonic power was controlled to 500 W, and the ultrasonic frequency was 20 kHz, then homogenized 3 times at 10 MPa for 10 min each time to form an emulsion, and a calcium chloride solution with a mass concentration of 20% was added, stirred at 400 r / min for 10 min, and then freeze-dried at -10°C for 10 h to obtain Chinese medicine microcapsules.

[0055] The bacterial enzyme solution in step 2) is prepared by the following method: according to the mass ratio of composite microorganisms, papain and water being 10:1:30, composite microorganisms (composed of a mixture of Lactobacillus acidophilus and Bacillus licheniformis with a live cell count ratio of 1:1.5), papain and water are stirred and mixed at a speed of 600 r / min for 30 minutes to obtain the bacterial enzyme solution.

[0056] Comparative Example 6 Comparative Example 6 is the same as Example 1, except that the raw material of the Chinese medicine microcapsule does not contain Gastrodia elata, as follows: The Chinese medicine microcapsule is prepared by the following method: 1) According to the mass ratio of Pueraria root, snow lotus and anhydrous ethanol of 5:1:50, Pueraria root and snow lotus are mixed, ground into powder, passed through a 100-mesh sieve, stirred with anhydrous ethanol at 600 r / min for 10 min, and then ultrasonically dispersed for 30 min under the control of ultrasonic frequency of 25 KHz and power of 500 W to obtain the primary extract; 2) Add the bacterial enzyme solution to the primary extract at a mass ratio of 1:0.3, stir at 500 r / min for 30 min, seal and ferment for 8 h, and then centrifuge at 10000 r / min for 30 min to obtain the Chinese medicine extract; 3) According to the mass ratio of Chinese medicine extract, chitosan and calcium chloride solution of 1:1:0.5, the Chinese medicine extract and chitosan were stirred at 400 r / min for 30 min, ultrasonically dispersed at 50°C for 10 min, the ultrasonic power was controlled to 500 W, and the ultrasonic frequency was 20 kHz, then homogenized 3 times at 10 MPa for 10 min each time to form an emulsion, and a calcium chloride solution with a mass concentration of 20% was added, stirred at 400 r / min for 10 min, and then freeze-dried at -10°C for 10 h to obtain Chinese medicine microcapsules.

[0057] The bacterial enzyme solution in step 2) is prepared by the following method: according to the mass ratio of composite microorganisms, papain and water being 10:1:30, composite microorganisms (composed of a mixture of Lactobacillus acidophilus and Bacillus licheniformis with a live cell count ratio of 1:1.5), papain and water are stirred and mixed at a speed of 600 r / min for 30 minutes to obtain the bacterial enzyme solution.

[0058] Comparative Example 7 Comparative Example 7 is the same as Example 1, except that the microorganisms in the bacterial enzyme solution are only Lactobacillus acidophilus, as follows: The Chinese medicine microcapsule is prepared by the following method: 1) Gastrodia elata, Pueraria root, Snow lotus and anhydrous ethanol were mixed in a mass ratio of 10:5:1:50, ground into powder, passed through a 100-mesh sieve, stirred with anhydrous ethanol at 600 r / min for 10 min, and then ultrasonically dispersed for 30 min under the control of ultrasonic frequency of 25 kHz and power of 500 W to obtain a primary extract; 2) Add the bacterial enzyme solution to the primary extract at a mass ratio of 1:0.3, stir at 500 r / min for 30 min, seal and ferment for 8 h, and then centrifuge at 10000 r / min for 30 min to obtain the Chinese medicine extract; 3) According to the mass ratio of Chinese medicine extract, chitosan and calcium chloride solution of 1:1:0.5, the Chinese medicine extract and chitosan were stirred at 400 r / min for 30 min, ultrasonically dispersed at 50°C for 10 min, the ultrasonic power was controlled to 500 W, and the ultrasonic frequency was 20 kHz, then homogenized 3 times at 10 MPa for 10 min each time to form an emulsion, and a calcium chloride solution with a mass concentration of 20% was added, stirred at 400 r / min for 10 min, and then freeze-dried at -10°C for 10 h to obtain Chinese medicine microcapsules.

[0059] The bacterial enzyme solution in step 2) is prepared by the following method: according to the mass ratio of Lactobacillus acidophilus, papain and water being 10:1:30, Lactobacillus acidophilus, papain and water are stirred and mixed at a speed of 600 r / min for 30 minutes to obtain the bacterial enzyme solution.

[0060] Comparative Example 8 Comparative Example 8 is the same as Example 1, except that the microorganism in the bacterial enzyme solution is only Bacillus licheniformis, as follows: The Chinese medicine microcapsule is prepared by the following method: 1) Gastrodia elata, Pueraria root, Snow lotus and anhydrous ethanol were mixed in a mass ratio of 10:5:1:50, ground into powder, passed through a 100-mesh sieve, stirred with anhydrous ethanol at 600 r / min for 10 min, and then ultrasonically dispersed for 30 min under the control of ultrasonic frequency of 25 kHz and power of 500 W to obtain a primary extract; 2) Add the bacterial enzyme solution to the primary extract at a mass ratio of 1:0.3, stir at 500 r / min for 30 min, seal and ferment for 8 h, and then centrifuge at 10000 r / min for 30 min to obtain the Chinese medicine extract; 3) According to the mass ratio of Chinese medicine extract, chitosan and calcium chloride solution of 1:1:0.5, the Chinese medicine extract and chitosan were stirred at 400 r / min for 30 min, ultrasonically dispersed at 50°C for 10 min, the ultrasonic power was controlled to 500 W, and the ultrasonic frequency was 20 kHz, then homogenized 3 times at 10 MPa for 10 min each time to form an emulsion, and a calcium chloride solution with a mass concentration of 20% was added, stirred at 400 r / min for 10 min, and then freeze-dried at -10°C for 10 h to obtain Chinese medicine microcapsules.

[0061] The bacterial enzyme solution in step 2) is prepared by the following method: according to the mass ratio of Bacillus licheniformis, papain and water being 10:1:30, Bacillus licheniformis, papain and water are stirred and mixed at a speed of 600 r / min for 30 minutes to obtain the bacterial enzyme solution.

[0062] Comparative Example 9 Comparative Example 9 is the same as Example 1, except that only composite microbial fermentation and extraction are used, as follows: The Chinese medicine microcapsule is prepared by the following method: 1) Gastrodia elata, Pueraria root, Snow lotus and anhydrous ethanol were mixed in a mass ratio of 10:5:1:50, ground into powder, passed through a 100-mesh sieve, stirred with anhydrous ethanol at 600 r / min for 10 min, and then ultrasonically dispersed for 30 min under the control of ultrasonic frequency of 25 kHz and power of 500 W to obtain a primary extract; 2) Add the composite microorganisms to the primary extract at a mass ratio of 1:0.3, stir at 500 r / min for 30 min, seal and ferment for 8 h, and then centrifuge at 10000 r / min for 30 min to obtain the Chinese medicine extract; 3) According to the mass ratio of Chinese medicine extract, chitosan and calcium chloride solution of 1:1:0.5, the Chinese medicine extract and chitosan were stirred at 400 r / min for 30 min, ultrasonically dispersed at 50°C for 10 min, the ultrasonic power was controlled to 500 W, and the ultrasonic frequency was 20 kHz, then homogenized 3 times at 10 MPa for 10 min each time to form an emulsion, and a calcium chloride solution with a mass concentration of 20% was added, stirred at 400 r / min for 10 min, and then freeze-dried at -10°C for 10 h to obtain Chinese medicine microcapsules.

[0063] Among them, in step 2), the composite microorganisms are composed of a mixture of Lactobacillus acidophilus and Bacillus licheniformis with a live bacteria count ratio of 1:1.5.

[0064] Comparative Example 10 Comparative Example 10 is the same as Example 1, except that the Chinese medicine is made into powder, as follows: The Chinese medicine powder is prepared by the following method: 1) Gastrodia elata, Pueraria root, Snow lotus and anhydrous ethanol were mixed in a mass ratio of 10:5:1:50, ground into powder, passed through a 100-mesh sieve, stirred with anhydrous ethanol at 600 r / min for 10 min, and then ultrasonically dispersed for 30 min under the control of ultrasonic frequency of 25 kHz and power of 500 W to obtain a primary extract; 2) Add the bacterial enzyme solution to the primary extract at a mass ratio of 1:0.3, stir at 500 r / min for 30 min, seal and ferment for 8 h, and then centrifuge at 10000 r / min for 30 min to obtain the Chinese medicine extract; 3) Freeze-dry the Chinese medicine extract at -10°C for 10 h to obtain Chinese medicine powder.

[0065] The bacterial enzyme solution in step 2) is prepared by the following method: according to the mass ratio of composite microorganisms, papain and water being 10:1:30, composite microorganisms (composed of a mixture of Lactobacillus acidophilus and Bacillus licheniformis with a live cell count ratio of 1:1.5), papain and water are stirred and mixed at a speed of 600 r / min for 30 minutes to obtain the bacterial enzyme solution.

[0066] Performance Testing The effects of the stem cell preparations prepared in Examples 1-5 of the present application and Comparative Examples 1-10 on treating cerebral stroke were tested respectively.

[0067] 1. Toxicity testing of functional additives The umbilical cord mesenchymal stem cells cultured to the fourth generation after recovery were digested with trypsin, and then the digestion was terminated by adding DMEM medium containing 10% fetal bovine serum. After the culture medium was centrifuged, new DMEM medium containing 10% fetal bovine serum was added to resuspend the cells to prepare a cell suspension; 100 μl (1×10 4 The cell suspension was inoculated into a 96-well plate, mixed by the cross method, and cultured at 37°C and 5% CO2 saturated humidity for 24 h. Normal control group: replaced with new DMEM medium (without fetal bovine serum); 20 μM functional aid group: replaced with functional aid medium with a final concentration of 20 μM (DMEM medium configuration without fetal bovine serum); 50 μM functional aid group: replaced with functional aid medium with a final concentration of 50 μM (DMEM medium configuration without fetal bovine serum); 100 μM functional aid group: replaced with functional aid medium with a final concentration of 100 μM (DMEM medium without fetal bovine serum); Blank control group: Add new DMEM medium (without fetal bovine serum) to the blank wells.

[0068] After the treatment, the cells were placed at 37°C and 5% CO2 saturated humidity for 48 h. After the culture, 10 μl of MTT was added to each well and incubated at 37°C for 4 h. The culture medium was removed, 150 μl of DMSO was added and shaken for 10 min, and the absorbance OD of each well was measured by an enzyme-labeled instrument. 570 , the cell viability of each group was detected, cell survival rate (%) = (OD experimental group / OD blank group) × 100%, the results are shown in Table 1. Table 1

[0069] According to Table 1, compared with the normal control group, there is no significant difference in cell survival rate between the functional adjuvant groups with different concentrations and the control group, indicating that the functional adjuvant of the present application does not produce obvious toxic effects on umbilical cord mesenchymal stem cells.

[0070] 2. Mcao model preparation The Mcao model was prepared using a modified suture method. 250-280g SD male rats were selected. They were fasted but not watered for 12h before surgery and anesthetized with 2% sodium pentobarbital 0.2ml / 100g intraperitoneally. The anesthetized rats were placed supine on a mouse board, with their limbs and head fixed and placed on the operating table. The hair near the neck was shaved, and routine skin preparation and disinfection were performed. During the operation, an incision of about 0.8cm was first made on the right side of the middle of the neck, and then the subcutaneous tissue was separated using a blunt tool to expose the internal carotid artery (ICA), external carotid artery (ECA), and common carotid artery (CCA). When separating the common carotid artery, special attention should be paid to the position of the vagus nerve. After the separation operation is completed, the common carotid artery and external carotid artery are ligated, and the internal carotid artery is closed by using an artery clamp. Next, a suture is introduced into the internal carotid artery through a small incision, and a marking point is placed at the bifurcation of the blood vessel to block the blood flow of the right middle cerebral artery. Finally, the suture was performed layer by layer, the suture incision was disinfected, and the blood was removed to prevent the rats from biting each other. In the sham group, the blood vessels were only separated without inserting the thread plug. After surgery, the rats were placed on a heating pad or covered with feed in a single cage. Two hours after the thread plug was inserted, the thread plug was pulled out 1 cm to cause the reperfusion process. After the operation, the successful modeling was judged by paralysis of the left limbs, unstable standing, and turning to one side after waking up from anesthesia.

[0071] 3. Drug administration and grouping The experiment required 136 SD male rats, and a total of 17 groups were set up: Sham group (sham operation), Model group (model group), and stem cell preparation treatment group. After 24 hours of perfusion, the stem cell preparation treatment group was given the stem cell preparation prepared in Examples 1-5 and Comparative Examples 1-10 for treatment, injected once a day, with a dose of 100 μl / rat, for 14 days. The Sham group and the Model group were given an equal amount of distilled water every day.

[0072] 4. Neurological function score The drug efficacy was monitored for 14 days, and the mNSS neurological function scores were performed on the 1st, 7th, and 14th days of administration. The Longa scoring method was used as a reference, with a total of 5 points: 0 points, no symptoms of neurological injury; 1 point, the contralateral forepaw could not be fully extended; 2 points, turning in circles to the contralateral side; 3 points, falling to the contralateral side; 4 points, unable to walk spontaneously, and loss of consciousness. The higher the score, the more severe the neurobehavioral disorder of the animal. The scoring results are shown in Table 2.

[0073] Table 2

[0074] Note: * <0.05 (vs Sham group), P # <0.05(vsMcao group).

[0075] According to the neurological function scores of rats, rats in the Sham group were not treated with plugging, and they showed normal behavior, no neurological damage, and a score of 0. In contrast, the neurological function scores of rats in the other groups were significantly improved after ischemia-reperfusion injury, indicating that the model was successfully prepared. Compared with the Model group, the scores of rats treated with the stem cell preparations prepared in Examples 1-5 of the present application and Comparative Examples 1-10 decreased significantly, indicating that the nerve damage of rats was significantly alleviated after 14 days of treatment, and the therapeutic effect of Examples 1-5 of the present application was significantly better than that of Comparative Examples 1-10, indicating that the stem cell preparations prepared in the present application can effectively alleviate ischemia-reperfusion nerve damage.

[0076] 5. Area of ​​cerebral infarction 14 days after the rats were administered, they were anesthetized with isoflurane, blood was collected from the abdominal aorta, and the blood was left to stand for 1 hour. The blood was centrifuged at 4°C and 4000 rpm for 10 minutes, and the supernatant (serum) was collected and stored at -80°C for later use. The rat brain was then quickly removed and placed in a -20°C refrigerator for 30 minutes. The olfactory bulb, cerebellum and brainstem were removed after removal, and average coronal sections were made. Six slices were cut with a thickness of about 2 mm, and then quickly placed in 2% red tetrazolium (TTC) staining solution for section staining. The reverse side was incubated at 37°C in the dark for 7 minutes, and the TTC solution was discarded after another 7 minutes of incubation. An appropriate amount of 4% paraformaldehyde was added, and the brain was fixed for 1 hour before observation and photography. The infarct area was calculated according to the following formula. The results are shown in Table 3.

[0077] Infarct area ratio = (area of ​​infarcted area / area of ​​the entire slice) × 100% Table 3

[0078] Note: * <0.05 (vs Sham group), P # <0.05(vsMcao group).

[0079] As can be seen from the data in Table 3, the Sham group is rats without sutures, and the infarction area is 0. Compared with the Model group, after treatment with the stem cell preparations prepared in Examples 1-5 of the present application and Comparative Examples 1-10, the cerebral infarction area of ​​the rats was significantly reduced, indicating that the stem cell preparations prepared in Examples 1-5 of the present application and Comparative Examples 1-10 can reduce the infarction area of ​​cerebral ischemia in Mcao rats, and the infarction area of ​​Example 1-5 is significantly lower than that of Comparative Examples 1-10, indicating that the stem cell preparations prepared in the present application have a good protective effect on cerebral ischemia-reperfusion.

[0080] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for preparing a stem cell preparation for treating cerebral stroke, characterized in that: The specific steps include: S1. Inoculate the optimized umbilical cord mesenchymal stem cells in serum-free medium and then culture them in an incubator. When the cell confluence reaches 70%-90%, digest and passage them with trypsin; S2, digesting the optimized umbilical cord mesenchymal stem cells passaged to the fourth generation with trypsin, adding DMEM medium containing fetal bovine serum to terminate the digestion, centrifuging the medium, adding new DMEM medium containing fetal bovine serum to resuspend the cells to prepare a cell suspension, and then diluting the cell suspension with physiological saline; S3. Add Chinese medicine microcapsules and sodium alginate to the diluted cell suspension, and centrifuge to obtain a stem cell preparation.

2. The method for preparing a stem cell preparation for treating cerebral stroke according to claim 1, characterized in that: In step S1, the optimized umbilical cord mesenchymal stem cells are prepared by the following method: (1) dissolving the functional additive in an ethanol solution and adding the solution to a serum-free culture medium to prepare a culture medium containing the functional additive; (2) Inoculate the umbilical cord mesenchymal stem cells into a culture vessel, and after the cells are completely attached to the wall, add a culture medium containing functional additives, and culture the cells for 2-4 days to obtain optimized umbilical cord mesenchymal stem cells.

3. The method for preparing a stem cell preparation for treating cerebral stroke according to claim 2, characterized in that: In the step (1), the functional auxiliary agent is composed of notoginseng total saponins, shikonin and catechins mixed in a mass ratio of 3-5:1:1.2-1.

6.

4. The method for preparing a stem cell preparation for treating cerebral stroke according to claim 1, characterized in that: In step S3, the Chinese medicine microcapsules are prepared by the following method: 1) Gastrodia elata, Pueraria root and snow lotus are mixed, ground into powder, sieved, stirred and mixed with anhydrous ethanol, and ultrasonically dispersed to obtain the primary extract; 2) Add bacterial enzyme solution to the primary extract, stir and mix, seal and ferment, and centrifuge to obtain the Chinese medicine extract; 3) The Chinese medicine extract and chitosan are stirred and mixed, ultrasonically dispersed, and then homogenized to form an emulsion, and calcium chloride solution is added, stirred and mixed, and then freeze-dried to obtain Chinese medicine microcapsules.

5. The method for preparing a stem cell preparation for treating cerebral stroke according to claim 4, characterized in that: In the step 1), the mass ratio of Gastrodia elata, Pueraria lobata, Saussurea involucrata and anhydrous ethanol is 10-14:5-7:1-3:

50.

6. The method for preparing a stem cell preparation for treating cerebral stroke according to claim 4, characterized in that: In the step 2), the mass ratio of the primary extract to the bacterial enzyme solution is 1:0.3-0.

7.

7. The method for preparing a stem cell preparation for treating cerebral stroke according to claim 6, characterized in that: The bacterial enzyme liquid is prepared by the following method: according to the mass ratio of composite microorganisms, papain and water being 10-12:1-3:30, the composite microorganisms, papain and water are stirred and mixed to obtain the bacterial enzyme liquid.

8. The method for preparing a stem cell preparation for treating cerebral stroke according to claim 7, characterized in that: The composite microorganism is composed of a mixture of Lactobacillus acidophilus and Bacillus licheniformis with a live bacteria count ratio of 1-1.4:1.5-1.

9.

9. The method for preparing a stem cell preparation for treating cerebral stroke according to claim 4, characterized in that: In the step 3), the mass ratio of the Chinese medicine extract, chitosan and calcium chloride solution is 1:1-3:0.5-0.

9.

10. A stem cell preparation for treating cerebral stroke, characterized in that: The stem cell preparation is prepared by the method for preparing a stem cell preparation for treating cerebral stroke according to any one of claims 1 to 9.

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