Stem cell preparation for treating Parkinson's disease and preparation method thereof

Abstract

The invention relates to a stem cell preparation for treating Parkinson's disease and a preparation method thereof, and belongs to the technical field of biological medicines.The preparation method comprises the following steps that a traditional Chinese medicine extract is encapsulated through solid lipid nanoparticles, ultrasonic treatment is conducted, and a compound substance is obtained; coating the compound with quaternized chitosan and hyaluronic acid, so as to obtain an encapsulated compound; and dispersing neural stem cells in a phosphate buffer solution to obtain a cell suspension, dispersing the encapsulated compound in deionized water to obtain a dispersion liquid, and adding the dispersion liquid into the cell suspension to obtain the stem cell preparation for treating Parkinson's disease. According to the technical scheme, the traditional Chinese medicine extract is doubly encapsulated by the solid lipid nanoparticles, the quaternized chitosan and the hyaluronic acid, so that active ingredients in the traditional Chinese medicine extract can be effectively protected, the biological activity and the stability of the traditional Chinese medicine extract are improved, proliferation and differentiation of stem cells are promoted, and the survival rate of the stem cells is increased. And the stem cell preparation can have a better effect in the aspect of preventing and treating Parkinson's disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a stem cell preparation for treating Parkinson's disease and a preparation method thereof. Background Art

[0002] Parkinson's disease, also known as "tremor paralysis," is one of the most common neurological diseases. Parkinson's disease occurs when cells that produce the neurotransmitter dopamine suddenly degenerate or their number decreases significantly in the substantia nigra region of the midbrain. The clinical manifestations of Parkinson's disease are mainly resting tremor, bradykinesia, limb rigidity, and postural balance disorders. At present, the treatment of Parkinson's disease is still mainly based on Western medicine, and the first choice of drug is levodopa. However, the efficacy of this drug decreases with the increase of medication time, and it causes a series of movement disorder complications, which causes certain harm to the patient's physical health. Therefore, it is necessary to find an effective therapy to inhibit or treat Parkinson's disease while reducing the side effects of taking levodopa.

[0003] Compared with conventional drug treatment, stem cell therapy has fewer side effects in inhibiting or treating Parkinson's disease, and can reduce patients' dependence on drugs and drug resistance. Stem cell therapy can improve the motor symptoms of Parkinson's patients to a certain extent, such as tremor, bradykinesia, muscle stiffness, etc. Although some patients have reported symptom improvement after receiving stem cell treatment, this effect is often temporary, and some patients may experience symptoms worsening again after a few months.

[0004] Stem cell preparations are prepared by mixing Chinese herbal extracts with stem cell suspensions. Chinese herbal extracts have good antioxidant effects and can enhance the efficacy of stem cell preparations. However, the active ingredients they contain may interact with stem cells and affect the proliferation and differentiation of stem cells. Therefore, it is still necessary to further enhance the stability of Chinese herbal extracts so that they can be successfully integrated into stem cells, thereby providing lasting drug efficacy and playing a better role in the treatment of Parkinson's disease. Summary of the invention

[0005] The purpose of the present invention is to provide a stem cell preparation for treating Parkinson's disease and a preparation method thereof. The Chinese medicine extract is encapsulated by solid lipid nanoparticles and subjected to ultrasonic treatment to obtain a composite substance. The Chinese medicine extract is composed of a mixture of resveratrol and pepper extract and is rich in multiple active ingredients. The solid lipid nanoparticles are used for encapsulation to effectively protect the active ingredients in the Chinese medicine extract, improve its stability and shelf life, and ultrasonic treatment improves the encapsulation efficiency of the Chinese medicine extract and enhances the stability of the system. The composite substance is coated with quaternized chitosan and hyaluronic acid to obtain an encapsulated composite. The composite substance is composed of a mixture of glycidyl trimethylammonium chloride and polyethylene glycol diglycidyl ether. The quaternary ammonium reagent can effectively improve the water solubility of chitosan, and the addition of hyaluronic acid can neutralize the positive charge in the quaternary ammonium chitosan. The composite material coated with the quaternary ammonium chitosan and hyaluronic acid not only improves the stability of the encapsulation system, but also can maintain the biological activity and stability of the active ingredients in the Chinese medicine extract; the neural stem cells are dispersed in a phosphate buffer to obtain a cell suspension, the encapsulation complex is dispersed in deionized water to obtain a dispersion, and the dispersion is added to the cell suspension to obtain a stem cell preparation for treating Parkinson's disease; the encapsulation complex is combined with the stem cells to effectively adsorb the stem cells, promote the proliferation and differentiation of the stem cells, and has a good effect in preventing and treating Parkinson's disease.

[0006] The technical problem to be solved by the present invention is: a stem cell preparation is prepared by mixing a traditional Chinese medicine extract with a stem cell suspension. The traditional Chinese medicine extract has a good antioxidant effect and can enhance the drug efficacy of the stem cell preparation, but the active ingredients contained therein may interact with the stem cells and affect the proliferation and differentiation of the stem cells. Therefore, it is still necessary to further enhance the stability of the traditional Chinese medicine extract so that it can be successfully integrated into the stem cells, thereby providing lasting drug efficacy and playing a better role in the treatment of Parkinson's disease.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] A method for preparing a stem cell preparation for treating Parkinson's disease comprises the following steps:

[0009] S1: Encapsulating the Chinese herbal medicine extract with solid lipid nanoparticles and performing ultrasonic treatment to obtain a composite substance;

[0010] S2: coating the composite material in step S1 with quaternized chitosan and hyaluronic acid to obtain an encapsulated composite;

[0011] S3: Disperse the neural stem cells in phosphate buffer to obtain a cell suspension, disperse the encapsulation complex in step S2 in deionized water to obtain a dispersion, homogenize and sterilize by vacuum filtration, then add the dispersion to the cell suspension and blow evenly to obtain a stem cell preparation for treating Parkinson's disease.

[0012] Furthermore, step S1 is specifically as follows:

[0013] The Chinese herbal medicine extract and solid lipid are dissolved in anhydrous ethanol at 35-45°C to obtain an organic phase, a stabilizer is added into deionized water, and then stirred at 65-75°C for 40-50 minutes to obtain an aqueous phase, the organic phase is added into the aqueous phase, and stirred at 65-75°C for 20-30 minutes, and then ultrasonically treated, and finally cooled at room temperature to obtain a composite substance.

[0014] Furthermore, the mass ratio of the traditional Chinese medicine extract, solid lipid and anhydrous ethanol is 0.08-0.12:0.3-0.4:25-30.

[0015] Furthermore, the solid lipid is composed of a mixture of palmitic acid and triglyceride in a mass ratio of 0.6-0.7:0.3-0.4.

[0016] Furthermore, the Chinese medicine extract is composed of resveratrol and pepper extract mixed in a mass ratio of 0.8-1:0.4-0.6.

[0017] Furthermore, the mass ratio of the stabilizer to deionized water is 0.15-0.25:200-220.

[0018] Furthermore, the stabilizer is composed of polyoxyethylene stearate and polysorbate 80 mixed in a mass ratio of 0.5-0.6:0.4-0.5.

[0019] Furthermore, the ultrasonic treatment time is 10-20 minutes, with an interval of 3-5 minutes to avoid overheating.

[0020] Furthermore, the preparation method of the pepper extract comprises the following steps:

[0021] The pepper is ground into fine powder, passed through a 300-400 mesh sieve, and then added into ethanol for extraction, stirred at 48-52° C. at a rotation speed of 300-400 rpm for 2-3 hours, filtered, and the ethanol is removed by rotary evaporation at 35-45° C. to finally obtain the pepper extract.

[0022] Furthermore, the mass ratio of the pepper to the ethanol is 0.8-1.2:15-20.

[0023] Furthermore, step S2 is specifically as follows:

[0024] The composite material in step S1 is dissolved in ethanol to obtain a composite solution, the composite solution is added to the chitosan solution and stirred for 10-20 minutes, then the hyaluronic acid solution is added, and stirring is continued for 20-25 minutes. After filtering, it is washed with deionized water and finally dried at room temperature to obtain an encapsulated complex.

[0025] In the above reaction process, the quaternized chitosan in the chitosan solution has an amino group, contains a quaternary ammonium salt and has a positive charge, and the hyaluronic acid in the hyaluronic acid solution has a carboxyl group. The amino group in the quaternized chitosan can combine with the carboxyl group in the hyaluronic acid and neutralize the positive charge on the quaternized chitosan, while encapsulating the composite substance in step S1, and finally obtaining an encapsulated composite.

[0026] Furthermore, the mass ratio of the composite substance to ethanol is 1-1.5:80-100.

[0027] Furthermore, the mass ratio of the composite solution to the chitosan solution is 1-2:7-8.

[0028] Furthermore, the chitosan solution is prepared by adding quaternized chitosan to deionized water at a mass ratio of 1-2:90-110.

[0029] Furthermore, the hyaluronic acid solution is prepared by adding hyaluronic acid into deionized water at a mass ratio of 1-1.5:80-100.

[0030] Furthermore, the preparation method of the quaternized chitosan comprises the following steps:

[0031] Add chitosan powder to deionized water, then add glacial acetic acid, and stir evenly, then add quaternary ammonium reagent, and stir for 25-35 minutes, and continue stirring at 50-60°C for 17-19 hours. After the reaction is completed, centrifuge at room temperature for 15-20 minutes, collect the supernatant, filter, dialyze with deionized water for 3-5 days, and freeze-dry at -20--15°C to obtain quaternary ammonium chitosan.

[0032] In the above reaction process, chitosan has amino groups and the quaternary ammonium reagent has epoxy groups. The amino groups in chitosan can combine with the epoxy groups in the quaternary ammonium reagent to graft the quaternary ammonium reagent onto chitosan, and finally obtain quaternary ammonium chitosan.

[0033] Furthermore, the mass ratio of the chitosan powder, deionized water, glacial acetic acid and quaternary ammonium reagent is 0.9-1.1:35-40:0.1-0.2:1.4-1.6.

[0034] Furthermore, the quaternary ammonium reagent is composed of a mixture of glycidyl trimethyl ammonium chloride and polyethylene glycol diglycidyl ether in a mass ratio of 0.6-0.7:0.3-0.4.

[0035] Furthermore, in step S3, the density of neural stem cells in the cell suspension is 2×10 6 -3×10 6 Pieces / mL.

[0036] Furthermore, in step S3, the mass ratio of the encapsulation complex to deionized water is 1-2:100-150.

[0037] Furthermore, in step S3, the homogenization process is carried out at a speed of 1000-1500 r / min for 10-20 s.

[0038] Furthermore, in step S3, the volume ratio of the dispersion liquid to the cell suspension is 95-97:3-5.

[0039] Beneficial effects of the present invention:

[0040] (1) In the technical scheme of the present invention, a Chinese medicine extract is encapsulated by solid lipid nanoparticles and subjected to ultrasonic treatment to obtain a composite substance; the Chinese medicine extract is composed of a mixture of resveratrol and pepper extract, and the pepper extract is rich in various active ingredients, such as capsaicin, etc. Capsaicin has anti-cancer, analgesic, antioxidant, antibacterial and other characteristics, and can increase the number of dopamine neurons in the substantia nigra area, transform microglia into an anti-inflammatory state, help prevent and manage neurodegenerative diseases, and protect the brain from free radical damage; resveratrol is a natural polyphenol flavonoid, which also has good anti-cancer, anti-inflammatory and brain protection effects, and can protect neurons from oxidative damage and toxicity and prevent apoptotic neuronal death; the active ingredients in resveratrol and pepper extract, under high temperature and pH value, The active ingredients in the Chinese herbal extracts are easily degraded or inactivated under conditions such as changes in temperature and ultraviolet rays. Encapsulation through solid lipid nanoparticles can effectively protect the active ingredients in the Chinese herbal extracts, improve their stability and shelf life, and can bypass liver and spleen filtration, prolong blood circulation, and improve the bioavailability of the Chinese herbal extracts. The solid lipid nanoparticles are formed by mixing solid lipids and stabilizers, including solid lipids composed of a mixture of palmitic acid and triglycerides and stabilizers composed of a mixture of polyoxyethylene stearate and polysorbate 80. The solid lipid nanoparticles have good encapsulation efficiency, and through ultrasonic treatment, the size of the nanoparticles can be further reduced, the encapsulation efficiency of the Chinese herbal extracts can be improved, the agglomeration phenomenon can be reduced, the stability of the system can be enhanced, and the efficacy of the Chinese herbal extracts in the treatment of Parkinson's disease can be improved.

[0041] (2) In the technical scheme of the present invention, the composite material is coated with quaternized chitosan and hyaluronic acid to obtain an encapsulated composite material; the quaternized chitosan is obtained by combining a quaternary ammonium reagent with chitosan. The water solubility of chitosan is poor. The quaternary ammonium reagent composed of a mixture of glycidyl trimethyl ammonium chloride and polyethylene glycol diglycidyl ether can effectively improve the water solubility of chitosan. By adding hyaluronic acid, the positive charge in the quaternized chitosan can be neutralized, thereby improving its biocompatibility and reducing cytotoxicity. Coating the composite material with quaternized chitosan and hyaluronic acid not only improves the stability of the encapsulation system, but also enables the active ingredients in the Chinese medicine extract to maintain their biological activity and stability. The effect of active ingredients in Chinese herbal extracts on the proliferation and differentiation of stem cells can be avoided, which is more conducive to the adsorption of stem cells on the encapsulation complex, thereby improving the drug efficacy of stem cell preparations and being beneficial to the prevention and treatment of Parkinson's disease; neural stem cells are dispersed in phosphate buffer to obtain a cell suspension, the encapsulation complex is dispersed in deionized water to obtain a dispersion, and after homogenization and vacuum filtration sterilization, the dispersion is added to the cell suspension and blown evenly to obtain a stem cell preparation for the treatment of Parkinson's disease; the encapsulation complex is combined with the stem cells, thereby enhancing its adsorption to the stem cells, promoting the proliferation and differentiation of the stem cells, and helping the stem cell preparations to achieve better results in the prevention and treatment of Parkinson's disease.

[0042] (3) In the technical scheme of the present invention, the Chinese herbal medicine extract is encapsulated by solid lipid nanoparticles, and then ultrasonically treated, and then coated with quaternary aminated chitosan and hyaluronic acid, and the encapsulated complex is dispersed in deionized water to obtain a dispersion, which is then added to a cell suspension to finally obtain a stem cell preparation for the treatment of Parkinson's disease; the double encapsulation of the Chinese herbal medicine extract by solid lipid nanoparticles and quaternary aminated chitosan and hyaluronic acid can not only effectively improve the activity and stability of the Chinese herbal medicine extract, promote the absorption and utilization of the active substances in the Chinese herbal medicine extract in the body, but also the encapsulated complex can better adsorb stem cells, promote their proliferation and differentiation, provide more lasting drug efficacy, and play a better therapeutic effect in the prevention and treatment of Parkinson's disease. DETAILED DESCRIPTION

[0043] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] The specific parameters of the raw materials used in the present invention are as follows:

[0045] Neural stem cells, provided by Saiye (Guangzhou) Biotechnology Co., Ltd.; phosphate buffer, pH 6, sterile, provided by Shanghai Shangbao Biotechnology Co., Ltd.; resveratrol, CAS No.: 501-36-0, provided by Shanghai MacLean Biochemical Technology Co., Ltd.; pepper, variety AIG01 pepper, provided by Happy Biotech Co., Ltd. (Icheon, South Korea); chitosan, CAS No.: 9012-76-4, provided by Shanghai MacLean Biochemical Technology Co., Ltd.; hyaluronic acid, CAS No.: 9004-61-9, provided by Shanghai MacLean Biochemical Technology Co., Ltd.; palmitic acid, CAS No.: 57-10-3, provided by Shanghai Aladdin Biochemical Technology Co., Ltd.; polyoxyethylene stearate, CAS No.: 9005-00-9, provided by Shanghai MacLean Biochemical Technology Co., Ltd.; glycidyl trimethylammonium chloride, CAS No.: 3033-77-0, provided by Shanghai Aladdin Biochemical Technology Co., Ltd.

[0046] Example 1

[0047] The method for preparing a stem cell preparation for treating Parkinson's disease comprises the following steps:

[0048] S1: Preparation of composite material, the specific steps are:

[0049] According to the mass ratio of Chinese herbal medicine extract, solid lipid and anhydrous ethanol of 0.08:0.3:25, the Chinese herbal medicine extract and solid lipid were dissolved in anhydrous ethanol at 35°C to obtain an organic phase. According to the mass ratio of stabilizer to deionized water of 0.15:200, the stabilizer was added into deionized water, and then stirred at 65°C for 40 min to obtain an aqueous phase. According to the mass ratio of organic phase to aqueous phase of 2:1, the organic phase was added into the aqueous phase, and stirred at 65°C at a speed of 300 rpm for 20 min to completely remove the After removing the anhydrous ethanol, the mixture was ultrasonically treated for 10 minutes (ultrasonic power of 100 W and ultrasonic frequency of 40 kHz) with an interval of 3 minutes to avoid overheating, and finally cooled at room temperature to obtain a composite material, wherein the solid lipid was composed of palmitic acid and triglyceride mixed in a mass ratio of 0.6:0.3; the Chinese medicine extract was composed of resveratrol and pepper extract mixed in a mass ratio of 0.8:0.4; the stabilizer was composed of polyoxyethylene stearate and polysorbate 80 mixed in a mass ratio of 0.5:0.4;

[0050] The preparation method of the pepper extract comprises the following steps:

[0051] According to the mass ratio of pepper to ethanol of 0.8:15, the pepper was ground into fine powder, passed through a 300-mesh sieve, and then added into ethanol for extraction, stirred at 48°C at a speed of 300 rpm for 2 hours, filtered, and the ethanol was removed by rotary evaporation at 35°C to obtain the pepper extract;

[0052] S2: Prepare encapsulation complex, the specific steps are:

[0053] According to the mass ratio of the composite substance to ethanol being 1:80, the composite substance in step S1 is dissolved in ethanol to obtain a composite solution, according to the mass ratio of the composite solution to the chitosan solution being 1:7, the composite solution is added to the chitosan solution, and stirred for 10 minutes, and then a hyaluronic acid solution is added (the mass of the hyaluronic acid solution is 2 times the mass of the composite solution), and the stirring is continued for 20 minutes. After filtering, the mixture is washed with deionized water (the mass of the deionized water is 20wt% of the mass of the ethanol), and finally dried at room temperature to obtain an encapsulation composite, wherein the chitosan solution is prepared by adding quaternized chitosan to deionized water at a mass ratio of 1:90; the hyaluronic acid solution is prepared by adding hyaluronic acid to deionized water at a mass ratio of 1:80;

[0054] The preparation method of quaternized chitosan comprises the following steps:

[0055] According to the mass ratio of chitosan powder, deionized water, glacial acetic acid and quaternary ammonium reagent being 0.9:35:0.1:1.4, chitosan powder is added into deionized water, and then 17.5 mol / L glacial acetic acid is added and stirred evenly, and then the quaternary ammonium reagent is added and stirred for 25 minutes, and the stirring is continued for 17 hours at 50°C. After the reaction is completed, centrifugation is performed at room temperature at a speed of 4000 rpm for 15 minutes, and the supernatant is collected. After filtration, it is dialyzed with deionized water for 3 days, and freeze-dried at minus 20°C to obtain quaternary ammonium chitosan, wherein the quaternary ammonium reagent is composed of glycidyl trimethylammonium chloride and polyethylene glycol diglycidyl ether mixed in a mass ratio of 0.6:0.3;

[0056] S3: Preparation of stem cell preparation for the treatment of Parkinson's disease, the specific steps are:

[0057] The neural stem cells were dispersed in phosphate buffer to obtain a cell suspension with a volume of 5 mL. The encapsulation complex in step S2 was dispersed in deionized water at a mass ratio of 1:100 to obtain a dispersion. The dispersion was homogenized at a speed of 1000 r / min for 20 s. After vacuum filtration sterilization (the vacuum degree of vacuum filtration was 50 KPa), the dispersion was added to the cell suspension at a volume ratio of 95:3 to the cell suspension, and the dispersion was blown evenly to obtain a stem cell preparation for treating Parkinson's disease. In the cell suspension, the density of the neural stem cells was 2×106 Pieces / mL.

[0058] Example 2

[0059] The method for preparing a stem cell preparation for treating Parkinson's disease comprises the following steps:

[0060] S1: Preparation of composite material, the specific steps are:

[0061] According to the mass ratio of Chinese herbal medicine extract, solid lipid and anhydrous ethanol of 0.1:0.35:28, the Chinese herbal medicine extract and solid lipid were dissolved in anhydrous ethanol at 40°C to obtain an organic phase. According to the mass ratio of stabilizer to deionized water of 0.2:210, the stabilizer was added into deionized water, and then stirred at 70°C for 45 minutes to obtain an aqueous phase. According to the mass ratio of organic phase to aqueous phase of 2.5:1.5, the organic phase was added into the aqueous phase, and stirred at 70°C at a speed of 400 rpm for 25 minutes to completely remove anhydrous ethanol, and then ultrasonically treated for 15 minutes (ultrasonic power of 100 W, ultrasonic frequency of 40 kHz), with an interval of 4 minutes to avoid overheating, and finally cooled at room temperature to obtain a composite material, wherein the solid lipid is composed of palmitic acid and triglyceride mixed in a mass ratio of 0.65:0.35; the Chinese medicine extract is composed of resveratrol and pepper extract mixed in a mass ratio of 0.9:0.5; the stabilizer is composed of polyoxyethylene stearate and polysorbate 80 mixed in a mass ratio of 0.55:0.45;

[0062] The preparation method of the pepper extract comprises the following steps:

[0063] According to the mass ratio of pepper to ethanol being 1:18, the pepper was ground into fine powder, passed through a 350-mesh sieve, and then added into ethanol for extraction, stirred at 50°C and 350 rpm for 2.5 h, filtered, and the ethanol was removed by rotary evaporation at 40°C to obtain the pepper extract;

[0064] S2: Prepare encapsulation complex, the specific steps are:

[0065] According to the mass ratio of the composite substance to ethanol being 1.2:90, the composite substance in step S1 is dissolved in ethanol to obtain a composite solution, according to the mass ratio of the composite solution to the chitosan solution being 1.5:7.5, the composite solution is added to the chitosan solution, and stirred for 15 minutes, and then a hyaluronic acid solution is added (the mass of the hyaluronic acid solution is twice the mass of the composite solution), and stirring is continued for 23 minutes. After filtering, the mixture is washed with deionized water (the mass of the deionized water is 20wt% of the mass of the ethanol), and finally dried at room temperature to obtain an encapsulation composite, wherein the chitosan solution is prepared by adding quaternized chitosan to deionized water at a mass ratio of 1.5:100; the hyaluronic acid solution is prepared by adding hyaluronic acid to deionized water at a mass ratio of 1.2:90;

[0066] The preparation method of quaternized chitosan comprises the following steps:

[0067] According to the mass ratio of chitosan powder, deionized water, glacial acetic acid and quaternary ammonium reagent being 1:38:0.15:1.5, chitosan powder is added into deionized water, and then 17.5 mol / L glacial acetic acid is added and stirred evenly, and then the quaternary ammonium reagent is added and stirred for 30 minutes, and the stirring is continued at 55°C for 18 hours. After the reaction is completed, centrifugation is performed at a speed of 4500 rpm for 18 minutes at room temperature, and the supernatant is collected. After filtration, it is dialyzed with deionized water for 4 days, and freeze-dried at minus 18°C ​​to obtain quaternary ammonium chitosan, wherein the quaternary ammonium reagent is composed of glycidyl trimethylammonium chloride and polyethylene glycol diglycidyl ether mixed in a mass ratio of 0.65:0.35;

[0068] S3: Preparation of stem cell preparation for the treatment of Parkinson's disease, the specific steps are:

[0069] The neural stem cells were dispersed in phosphate buffer to obtain a cell suspension with a volume of 5 mL. The encapsulation complex in step S2 was dispersed in deionized water at a mass ratio of 1.5:120 to obtain a dispersion. The dispersion was homogenized at a speed of 1200 r / min for 15 s. After vacuum filtration sterilization (the vacuum degree of vacuum filtration was 53 KPa), the dispersion was added to the cell suspension at a volume ratio of 96:4 to the cell suspension, and the dispersion was blown evenly to obtain a stem cell preparation for treating Parkinson's disease. In the cell suspension, the density of the neural stem cells was 2.5×10 6 Pieces / mL.

[0070] Example 3

[0071] The method for preparing a stem cell preparation for treating Parkinson's disease comprises the following steps:

[0072] S1: Preparation of composite material, the specific steps are:

[0073] According to the mass ratio of Chinese herbal medicine extract, solid lipid and anhydrous ethanol of 0.12:0.4:30, the Chinese herbal medicine extract and solid lipid were dissolved in anhydrous ethanol at 45°C to obtain an organic phase. According to the mass ratio of stabilizer to deionized water of 0.25:220, the stabilizer was added into deionized water, and then stirred at 75°C for 50 minutes to obtain an aqueous phase. According to the mass ratio of organic phase to aqueous phase of 3:2, the organic phase was added into the aqueous phase, and stirred at 75°C at a speed of 500 rpm for 30 minutes to completely The anhydrous ethanol was removed, and then ultrasonic treatment was performed for 20 minutes (ultrasonic power of 100 W, ultrasonic frequency of 40 kHz), with an interval of 5 minutes to avoid overheating, and finally cooled at room temperature to obtain a composite material, wherein the solid lipid was composed of palmitic acid and triglyceride mixed in a mass ratio of 0.7:0.4; the Chinese medicine extract was composed of resveratrol and pepper extract mixed in a mass ratio of 1:0.6; the stabilizer was composed of polyoxyethylene stearate and polysorbate 80 mixed in a mass ratio of 0.6:0.5;

[0074] The preparation method of the pepper extract comprises the following steps:

[0075] According to the mass ratio of pepper to ethanol of 1.2:20, the pepper was ground into fine powder, passed through a 400-mesh sieve, and then added into ethanol for extraction, stirred at 400 rpm at 52°C for 3 h, filtered, and the ethanol was removed by rotary evaporation at 45°C to obtain the pepper extract;

[0076] S2: Prepare encapsulation complex, the specific steps are:

[0077] According to the mass ratio of the composite substance to ethanol being 1.5:100, the composite substance in step S1 is dissolved in ethanol to obtain a composite solution, according to the mass ratio of the composite solution to the chitosan solution being 2:8, the composite solution is added to the chitosan solution, and stirred for 20 minutes, and then a hyaluronic acid solution is added (the mass of the hyaluronic acid solution is twice the mass of the composite solution), and the stirring is continued for 25 minutes. After filtering, the mixture is washed with deionized water (the mass of the deionized water is 20wt% of the mass of the ethanol), and finally dried at room temperature to obtain an encapsulation composite, wherein the chitosan solution is prepared by adding quaternized chitosan to deionized water at a mass ratio of 2:110; the hyaluronic acid solution is prepared by adding hyaluronic acid to deionized water at a mass ratio of 1.5:100;

[0078] The preparation method of quaternized chitosan comprises the following steps:

[0079] According to the mass ratio of chitosan powder, deionized water, glacial acetic acid and quaternary ammonium reagent being 1.1:40:0.2:1.6, chitosan powder is added into deionized water, and then 17.5 mol / L glacial acetic acid is added and stirred evenly, and then the quaternary ammonium reagent is added and stirred for 35 minutes, and the stirring is continued at 60°C for 19 hours. After the reaction is completed, centrifugation is performed at room temperature at a speed of 5000 rpm for 20 minutes, and the supernatant is collected. After filtration, it is dialyzed with deionized water for 5 days, and freeze-dried at minus 15°C to obtain quaternary ammonium chitosan, wherein the quaternary ammonium reagent is composed of glycidyl trimethylammonium chloride and polyethylene glycol diglycidyl ether mixed in a mass ratio of 0.7:0.4;

[0080] S3: Preparation of stem cell preparation for the treatment of Parkinson's disease, the specific steps are:

[0081] The neural stem cells were dispersed in phosphate buffer to obtain a cell suspension with a volume of 5 mL. The encapsulation complex in step S2 was dispersed in deionized water at a mass ratio of 2:150 to obtain a dispersion. The dispersion was homogenized at a speed of 1500 r / min for 10 s. After vacuum filtration sterilization (the vacuum degree of vacuum filtration was 55 KPa), the dispersion was added to the cell suspension at a volume ratio of 97:5 to the cell suspension, and the dispersion was blown evenly to obtain a stem cell preparation for treating Parkinson's disease. In the cell suspension, the density of the neural stem cells was 3×10 6 Pieces / mL.

[0082] Comparative Example 1

[0083] The difference between this comparative example and Example 3 is that when preparing the composite material, the mass of the Chinese herbal medicine extract is replaced by resveratrol, and the remaining steps and raw materials are the same as Example 3;

[0084] S1: Preparation of composite material, the specific steps are:

[0085] According to the mass ratio of resveratrol, solid lipid and anhydrous ethanol being 0.12:0.4:30, resveratrol and solid lipid are dissolved in anhydrous ethanol at 45°C to obtain an organic phase, according to the mass ratio of stabilizer to deionized water being 0.25:220, the stabilizer is added to deionized water, and then stirred at 75°C for 50 minutes to obtain an aqueous phase, according to the mass ratio of organic phase to aqueous phase being 3:2, the organic phase is added to the aqueous phase, and stirred at 75°C at a speed of 500 rpm for 30 minutes to completely remove anhydrous ethanol, and then ultrasonic treatment is performed for 20 minutes (ultrasonic power of 100 W, ultrasonic frequency of 40 kHz), with an interval of 5 minutes to avoid overheating, and finally cooled at room temperature to obtain a composite substance, wherein the solid lipid is composed of palmitic acid and triglyceride mixed in a mass ratio of 0.7:0.4; the stabilizer is composed of polyoxyethylene stearate and polysorbate 80 mixed in a mass ratio of 0.6:0.5.

[0086] Comparative Example 2

[0087] The difference between this comparative example and Example 3 is that when preparing the composite material, the solid lipid is replaced by palmitic acid, and the remaining steps and raw materials are the same as those of Example 3;

[0088] S1: Preparation of composite material, the specific steps are:

[0089] According to the mass ratio of Chinese herbal medicine extract, palmitic acid and anhydrous ethanol being 0.12:0.4:30, the Chinese herbal medicine extract and palmitic acid are dissolved in anhydrous ethanol at 45°C to obtain an organic phase, according to the mass ratio of stabilizer to deionized water being 0.25:220, the stabilizer is added to deionized water, and then stirred at 75°C for 50 minutes to obtain an aqueous phase, according to the mass ratio of organic phase to aqueous phase being 3:2, the organic phase is added to the aqueous phase, and stirred at 75°C at a speed of 500 rpm for 30 minutes to completely remove anhydrous ethanol, and then ultrasonic treatment is performed for 20 minutes (ultrasonic power is 100 W, ultrasonic frequency is 40 kHz), with an interval of 5 minutes to avoid overheating, and finally cooled at room temperature to obtain a composite substance, wherein the Chinese herbal medicine extract is composed of resveratrol and pepper extract mixed in a mass ratio of 1:0.6; the stabilizer is composed of polyoxyethylene stearate and polysorbate 80 mixed in a mass ratio of 0.6:0.5.

[0090] Comparative Example 3

[0091] The difference between this comparative example and Example 3 is that when preparing the composite material, the stabilizer and other materials are replaced by polyoxyethylene stearate, and the remaining steps and raw materials are synchronized with Example 3;

[0092] S1: Preparation of composite material, the specific steps are:

[0093] According to the mass ratio of Chinese herbal medicine extract, solid lipid and anhydrous ethanol being 0.12:0.4:30, the Chinese herbal medicine extract and solid lipid are dissolved in anhydrous ethanol at 45°C to obtain an organic phase, according to the mass ratio of polyoxyethylene stearate to deionized water being 0.25:220, polyoxyethylene stearate is added to deionized water, and then stirred at 75°C for 50 minutes to obtain an aqueous phase, according to the mass ratio of organic phase to aqueous phase being 3:2, the organic phase is added to the aqueous phase, and stirred at 75°C at a speed of 500 rpm for 30 minutes to completely remove anhydrous ethanol, and then ultrasonic treatment is performed for 20 minutes (ultrasonic power is 100 W, ultrasonic frequency is 40 kHz), with an interval of 5 minutes to avoid overheating, and finally cooled at room temperature to obtain a composite substance, wherein the solid lipid is composed of palmitic acid and triglyceride mixed in a mass ratio of 0.7:0.4; the Chinese herbal medicine extract is composed of resveratrol and pepper extract mixed in a mass ratio of 1:0.6.

[0094] Comparative Example 4

[0095] The difference between this comparative example and Example 3 is that when preparing quaternized chitosan, the mass of the quaternizing agent is replaced by glycidyl trimethyl ammonium chloride, and the remaining steps and raw materials are the same as those of Example 3;

[0096] The preparation method of quaternized chitosan comprises the following steps:

[0097] According to the mass ratio of chitosan powder, deionized water, glacial acetic acid and glycidyl trimethyl ammonium chloride being 1.1:40:0.2:1.6, chitosan powder was added into deionized water, and then 17.5 mol / L glacial acetic acid was added and stirred evenly, and then glycidyl trimethyl ammonium chloride was added and stirred for 35 min, and continued to stir at 60°C for 19 h. After the reaction was completed, centrifuged at 5000 rpm for 20 min at room temperature, the supernatant was collected, filtered, dialyzed with deionized water for 5 days, and freeze-dried at minus 15°C to obtain quaternized chitosan.

[0098] Comparative Example 5

[0099] The difference between this comparative example and Example 3 is that when preparing the encapsulation complex, the mass of hyaluronic acid is replaced by gelatin, and the remaining steps and raw materials are the same as those of Example 3;

[0100] S2: Prepare encapsulation complex, the specific steps are:

[0101] According to the mass ratio of the composite substance to ethanol being 1.5:100, the composite substance in step S1 is dissolved in ethanol to obtain a composite solution, according to the mass ratio of the composite solution to the chitosan solution being 2:8, the composite solution is added to the chitosan solution, and stirred for 20 minutes, and then a gelatin solution is added (the mass of the gelatin solution is twice the mass of the composite solution), and stirring is continued for 25 minutes. After filtering, the mixture is washed with deionized water (the mass of the deionized water is 20wt% of the mass of the ethanol), and finally dried at room temperature to obtain an encapsulation composite, wherein the chitosan solution is prepared by adding quaternized chitosan to deionized water at a mass ratio of 2:110; and the gelatin solution is prepared by adding gelatin to deionized water at a mass ratio of 1.5:100.

[0102] Comparative Example 6

[0103] The difference between this comparative example and Example 3 is that when preparing the stem cell preparation for treating Parkinson's disease, the Chinese medicine extract is coated with quaternized chitosan and hyaluronic acid, the original step S1 is deleted, and the remaining steps and raw materials are synchronized with Example 3;

[0104] The method for preparing a stem cell preparation for treating Parkinson's disease comprises the following steps:

[0105] S1: Prepare encapsulation complex, the specific steps are:

[0106] According to the mass ratio of Chinese herbal medicine extract to ethanol of 1.5:100, the Chinese herbal medicine extract is dissolved in ethanol to obtain a composite solution, according to the mass ratio of the composite solution to chitosan solution of 2:8, the composite solution is added to the chitosan solution, and stirred for 20 minutes, and then a hyaluronic acid solution is added (the mass of the hyaluronic acid solution is twice the mass of the composite solution), and the stirring is continued for 25 minutes. After filtering, the mixture is washed with deionized water (the mass of deionized water is 20wt% of the mass of ethanol), and finally dried at room temperature to obtain an encapsulation complex, wherein the chitosan solution is prepared by adding quaternized chitosan to deionized water at a mass ratio of 2:110; the hyaluronic acid solution is prepared by adding hyaluronic acid to deionized water at a mass ratio of 1.5:100; the Chinese herbal medicine extract is composed of a mixture of resveratrol and pepper extract at a mass ratio of 1:0.6;

[0107] The preparation method of quaternized chitosan comprises the following steps:

[0108] According to the mass ratio of chitosan powder, deionized water, glacial acetic acid and quaternary ammonium reagent being 1.1:40:0.2:1.6, chitosan powder is added into deionized water, and then 17.5 mol / L glacial acetic acid is added and stirred evenly, and then the quaternary ammonium reagent is added and stirred for 35 minutes, and the stirring is continued at 60°C for 19 hours. After the reaction is completed, centrifugation is performed at room temperature at a speed of 5000 rpm for 20 minutes, and the supernatant is collected. After filtration, it is dialyzed with deionized water for 5 days, and freeze-dried at minus 15°C to obtain quaternary ammonium chitosan, wherein the quaternary ammonium reagent is composed of glycidyl trimethylammonium chloride and polyethylene glycol diglycidyl ether mixed in a mass ratio of 0.7:0.4;

[0109] S2: Preparation of stem cell preparation for the treatment of Parkinson's disease, the specific steps are:

[0110] The neural stem cells were dispersed in phosphate buffer to obtain a cell suspension with a volume of 5 mL. The encapsulation complex in step S2 was dispersed in deionized water at a mass ratio of 2:150 to obtain a dispersion. The dispersion was homogenized at a speed of 1500 r / min for 10 s. After vacuum filtration sterilization (the vacuum degree of vacuum filtration was 55 KPa), the dispersion was added to the cell suspension at a volume ratio of 97:5 to the cell suspension, and the dispersion was blown evenly to obtain a stem cell preparation for treating Parkinson's disease. In the cell suspension, the density of the neural stem cells was 3×10 6 Pieces / mL.

[0111] The Parkinson's disease treatment effects of the stem cell preparations prepared in Examples 1-3 and Comparative Examples 1-6 were tested.

[0112] (I) Animal model preparation

[0113] Two-month-old male C57BL / 6 mice (18±2g) were selected and adaptively fed for 1 week to establish the PD model. Model establishment method: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was injected intraperitoneally into the mice for 5 consecutive days (25 mg / kg / d).

[0114] (II) Main experimental drugs

[0115] The stem cell preparations for treating Parkinson's disease prepared in Examples 1-3 and Comparative Examples 1-6.

[0116] 3. Experimental groups and interventions

[0117] The mice were randomly divided into a normal group (i.e., blank control group), an MPTP model group, an MPTP+stem cell preparation group, a L-dopa group, and a stem cell group, with 8 mice in each group.

[0118] The mice in the MPTP model group, MPTP+stem cell preparation group, L-DOPA group, and stem cell group received intraperitoneal injection of MPTP (25 mg / kg / d) for 7 consecutive days; the mice in the normal group received intraperitoneal injection of the same volume of normal saline only for 7 consecutive days.

[0119] MPTP+stem cell preparation group: mice received intraperitoneal injection of stem cell preparations for treating Parkinson's disease prepared in Examples 1-3 and Comparative Examples 1-6, respectively, at a dose of 60 mg / kg, once a day, for 17 consecutive days; levodopa group: mice received intraperitoneal injection of the same dose of levodopa, once a day, for 17 consecutive days; stem cell group: mice received intraperitoneal injection of the same dose of neural stem cells, once a day, for 17 consecutive days; MPTP model group: mice received intraperitoneal injection of the same dose of normal saline, once a day, for 17 consecutive days; normal group (i.e., blank control group): mice received intraperitoneal injection of the same dose of normal saline, once a day, for 17 consecutive days.

[0120] (IV) Behavioral Experiments

[0121] 1. Open field experiment

[0122] The mice were placed in a 100cm×100cm square plastic frame with a camera installed above. The room was kept well-lit and quiet. After the adaptation period, the mice were allowed to move freely in the frame for 10 minutes. The relevant parameters were set using the SuperMaze Animal Behavior Video Analysis System (provided by Shanghai Xinruan Information Technology Co., Ltd.). The system tracked all the movement trajectories of the mice in the plastic frame and obtained the total movement distance of the mice. After each mouse test, the remaining excrement of the mice in the plastic frame should be cleaned with an alcohol cotton ball to avoid affecting the experimental results of the next mouse.

[0123] 2. Pole climbing experiment

[0124] First, a device was made by fixing a 2.5cm diameter cork ball on the top of a 50cm long and 1cm diameter wooden stick. Then, the wooden stick and the ball were wrapped with anti-slip tape, and the tail of the wooden stick was fixed so that the stick was perpendicular to the ground to make a simple climbing pole device. At the beginning of the experiment, the mouse was placed head down on the ball and allowed to climb down naturally. If it crawled in the opposite direction or paused halfway, it was retested and the total time taken by the mouse to climb from the top ball to the bottom of the wooden stick was recorded. Each mouse was tested 3 times, with an interval of more than 10 minutes each time, and the average value was taken for statistical analysis.

[0125] The test results are shown in Table 1 below:

[0126] Table 1 Behavioral experimental test results

[0127] project Total movement distance (mm) Climbing time(s) Example 1 38700 4.56±1.13 Example 2 39300 4.21±1.09 Example 3 38400 4.78±1.26 Comparative Example 1 32600 5.86±1.18 Comparative Example 2 35200 5.52±1.47 Comparative Example 3 34900 5.63±1.35 Comparative Example 4 34500 6.39±1.52 Comparative Example 5 33900 6.68±1.37 Comparative Example 6 31400 6.84±1.11 MPTP model group 22600 10.08±2.14 Blank control group 29900 7.28±1.76 Levodopa group 30300 7.02±1.04 Stem Cell Group 31000 6.91±1.23

[0128] It can be seen from the data in Table 1 above that, by comparing Comparative Examples 1-3 with Example 3, the mass of the Chinese medicine extract is replaced with resveratrol, or the mass of the solid lipid is replaced with palmitic acid, or the mass of the stabilizer is replaced with polyoxyethylene stearate, and finally a stem cell preparation for the treatment of Parkinson's disease is prepared. The test results are worse than those of Example 3, indicating that the Chinese medicine extract composed of a mixture of resveratrol and a pepper extract has good antioxidant, anti-inflammatory and neuroprotective effects, the solid lipid composed of a mixture of palmitic acid and triglycerides, the stabilizer composed of a mixture of polyoxyethylene stearate and polysorbate 80, and the solid lipid nanoparticles formed by the solid lipid and the stabilizer have good encapsulation efficiency, can effectively protect the active substances in the Chinese medicine extract, promote the absorption and utilization of the active substances in the body, and help the stem cell preparation to play a better role in the treatment of Parkinson's disease;

[0129] From the comparison between Comparative Examples 4-5 and Example 3, it can be seen that the quaternary ammonium reagent is replaced with glycidyl trimethyl ammonium chloride, or the hyaluronic acid is replaced with gelatin, and finally a stem cell preparation for treating Parkinson's disease is prepared. The test results are worse than those of Example 3, indicating that the quaternary ammonium reagent composed of a mixture of glycidyl trimethyl ammonium chloride and polyethylene glycol diglycidyl ether can better improve the water solubility and biocompatibility of chitosan. By adding hyaluronic acid, the positive charge on the quaternary ammonium chitosan can be neutralized. The composite material is encapsulated with quaternary ammonium chitosan and hyaluronic acid, which improves the encapsulation efficiency of the composite material, enhances the overall stability of the system, enables the active substances in the Chinese medicine extract to be fully absorbed and utilized in the body, and increases the proliferation and differentiation of stem cells, which is beneficial to the prevention and treatment of Parkinson's disease;

[0130] By comparing Comparative Example 6 with Example 3, it can be seen that the Chinese medicine extract is coated with quaternary aminated chitosan and hyaluronic acid, and finally a stem cell preparation for the treatment of Parkinson's disease is prepared. The test result is worse than that of Example 3, indicating that the Chinese medicine extract is first encapsulated by solid lipid nanoparticles, and then coated with quaternary aminated chitosan and hyaluronic acid. The double encapsulation of solid lipid nanoparticles and quaternary aminated chitosan and hyaluronic acid not only improves the overall stability of the encapsulation system, but also improves the stability and biological activity of the active substances in the Chinese medicine extract, promotes the absorption and utilization of the active substances in the body, and at the same time enhances the adsorption of the encapsulation complex on stem cells, promotes their proliferation and differentiation, and enables the stem cell preparation to play a better therapeutic effect in the treatment of Parkinson's disease.

[0131] It can be seen from the data in Table 1 above that the stem cell preparation for treating Parkinson's disease prepared in Example 1-3 is compared with the stem cell preparation for treating Parkinson's disease prepared in Comparative Example 1-6. The Chinese medicine extract is encapsulated by solid lipid nanoparticles, and then ultrasonically treated, and then coated with quaternized chitosan and hyaluronic acid, and the encapsulated complex is dispersed in deionized water to obtain a dispersion, which is then added to a cell suspension to finally obtain a stem cell preparation for treating Parkinson's disease, which meets the test performance requirements, while the stem cell preparation for treating Parkinson's disease prepared in Comparative Example 1-6 does not meet the performance requirements. The stem cell preparation for treating Parkinson's disease prepared by the present invention can effectively protect the active ingredients in the Chinese medicine extract, promote the absorption and utilization of the active substances in the Chinese medicine extract in the body, and increase the proliferation and differentiation of stem cells to provide a lasting drug efficacy, which helps the stem cell preparation to play a better role in the treatment of Parkinson's disease.

[0132] In the description of the specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0133] The above contents are merely examples and explanations of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the invention or exceed the scope defined by the claims, they shall all fall within the protection scope of the present invention.

Claims

1. A method for preparing a stem cell preparation for treating Parkinson's disease, characterized in that: The following steps are involved: S1: Encapsulating the Chinese herbal medicine extract with solid lipid nanoparticles and performing ultrasonic treatment to obtain a composite substance; S2: coating the composite material in step S1 with quaternized chitosan and hyaluronic acid to obtain an encapsulated composite; S3: Disperse the neural stem cells in phosphate buffer to obtain a cell suspension, disperse the encapsulation complex in step S2 in deionized water to obtain a dispersion, homogenize and sterilize by vacuum filtration, then add the dispersion to the cell suspension and blow evenly to obtain a stem cell preparation for treating Parkinson's disease.

2. The method for preparing a stem cell preparation for treating Parkinson's disease according to claim 1, characterized in that: Step S1 is specifically as follows: The Chinese herbal medicine extract and solid lipid are dissolved in anhydrous ethanol at 35-45°C to obtain an organic phase, a stabilizer is added into deionized water, and then stirred at 65-75°C for 40-50 minutes to obtain an aqueous phase, the organic phase is added into the aqueous phase, and stirred at 65-75°C for 20-30 minutes, and then ultrasonically treated, and finally cooled at room temperature to obtain a composite substance.

3. The method for preparing a stem cell preparation for treating Parkinson's disease according to claim 2, characterized in that: The solid lipid is composed of palmitic acid and triglyceride mixed in a mass ratio of 0.6-0.7:0.3-0.

4.

4. The method for preparing a stem cell preparation for treating Parkinson's disease according to claim 2, characterized in that: The traditional Chinese medicine extract is composed of resveratrol and pepper extract mixed in a mass ratio of 0.8-1:0.4-0.

6.

5. The method for preparing a stem cell preparation for treating Parkinson's disease according to claim 2, characterized in that: The stabilizer is composed of polyoxyethylene stearate and polysorbate 80 mixed in a mass ratio of 0.5-0.6:0.4-0.

5.

6. The method for preparing a stem cell preparation for treating Parkinson's disease according to claim 1, characterized in that: Step S2 is specifically as follows: The composite material in step S1 is dissolved in ethanol to obtain a composite solution, the composite solution is added to the chitosan solution and stirred for 10-20 minutes, then the hyaluronic acid solution is added, and stirring is continued for 20-25 minutes. After filtering, it is washed with deionized water and finally dried at room temperature to obtain an encapsulated complex.

7. The method for preparing a stem cell preparation for treating Parkinson's disease according to claim 1, characterized in that: The preparation method of the quaternized chitosan comprises the following steps: Add chitosan powder to deionized water, then add glacial acetic acid, and stir evenly, then add quaternary ammonium reagent, and stir for 25-35 minutes, and continue stirring at 50-60°C for 17-19 hours. After the reaction is completed, centrifuge at room temperature for 15-20 minutes, collect the supernatant, filter, dialyze with deionized water for 3-5 days, and freeze-dry at -20--15°C to obtain quaternary ammonium chitosan.

8. The method for preparing a stem cell preparation for treating Parkinson's disease according to claim 7, characterized in that: The quaternary ammonium reagent is composed of glycidyl trimethyl ammonium chloride and polyethylene glycol diglycidyl ether mixed in a mass ratio of 0.6-0.7:0.3-0.

4.

9. The method for preparing a stem cell preparation for treating Parkinson's disease according to claim 1, characterized in that: In step S3, the homogenization process is carried out at a speed of 1000-1500 r / min for 10-20 s.

10. A stem cell preparation for treating Parkinson's disease obtained by the preparation method according to any one of claims 1 to 9.