Culture method of genetically engineered cells for treating Parkinson's disease

Abstract

The invention discloses a culture method of genetically engineered cells for treating Parkinson's disease, which comprises the following steps of: synthesizing three required key genes, namely TH (Tyrosine Hydroxylase), ADDC (ADDC) and GCH1 (GTP-CycloHydroxy1), by using a dopamine neurotransmitter (DA), introducing the three key genes into mesenchymal stem cells (MSC) cultured in vitro by using a genetic engineering method, and enabling the MSC to express the three genes, so that the DA neurotransmitter is synthesized, and the DA neurotransmitter is used for treating the Parkinson's disease. And treating Parkinson's disease (PD) by transplanting these MSCs capable of synthesizing DA neurotransmitters to the corpus striatum of the brain. The MSC medicine for treating PD by exogenously supplementing dopamine neurotransmitter prepared by the invention has the characteristics of low cost, high activity, low immunogenicity and obvious treatment effect. The medicine prepared from the human MSC does not generate the medicine side effect of symptom fluctuation, can continuously survive in a host body for a long time and generate activity, and provides an experimental basis and a theoretical basis for establishing a PD treatment strategy and designing a novel stem cell treatment medicine.

Description

A method for culturing genetically engineered cells for treating Parkinson's disease Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a method for culturing genetically engineered cells for treating Parkinson's disease. Background Art

[0002] Parkinson's disease (PD) is the second most common degenerative disorder of the central nervous system worldwide. It is caused by the gradual loss of dopaminergic (DA) neurons in the midbrain. Essentially, this is due to a decrease in dopamine neurotransmitter production by DA neurons, leading to a breakdown of the nigrostriatal dopamine circuit. Dopamine supplementation is an effective strategy for treating PD. Treatment of PD has long relied on oral levodopa, which is particularly effective in the early stages of Parkinson's disease. However, long-term systemic administration and the eventual death of dopamine neurons make oral levodopa ineffective or produce significant side effects.

[0003] Dopamine (DOPA) is synthesized from L-tyrosine in two steps: first, tyrosine hydroxylase (TH) hydroxylates L-tyrosine to L-DOPA (L-3,4-dihydroxyphenylalanine), which is then converted to DOPA by aromatic L-amino acid decarboxylase (AADC). TH requires tetrahydrobiopterin (BH4) as a cofactor for its catalytic activity, which is then catalyzed by GTP-cyclohydrolase-1 (GCH1) to generate GTP. Furthermore, mesenchymal stem cells (MSCs) are readily available, possess multipotential differentiation, strong self-renewal capacity, low immunogenicity, a high safety profile, and the ability to regulate inflammatory responses, possessing properties that protect against and promote neuroregeneration. Therefore, they are used to ameliorate neurodegenerative diseases and are an important donor cell for the treatment of Parkinson's disease.

[0004] Through genetic engineering, this vector contains genes encoding all three enzymes required for efficient dopamine synthesis (TH, AADC, and GCH1). This vector was then infected with MSCs to create a stably infected cell line, termed MSC-DOPA. This cell line can stably secrete the neurotransmitter DOPA. Transplantation of MSC-DOPA into a 6-OHDA-induced PD rat model and an MPP+-induced PD monkey model replenished dopamine neurotransmitter loss and significantly improved behavioral and motor function in both PD models, achieving the goal of treating PD.

[0005] Therefore, this paper proposes a method for culturing genetically engineered cells for treating Parkinson's disease.

[0006] Summary of the Invention

[0007] To address the above-mentioned technical problems, the present invention has designed a method for culturing genetically engineered cells for treating Parkinson's disease. The MSC drug prepared by the present invention, which can exogenously supplement dopamine neurotransmitters to treat PD, has the characteristics of low cost, high activity, low immunogenicity, and significant therapeutic effect. The human MSC-DOPA produced by the present invention does not produce the drug side effect of fluctuating PD symptoms, and can survive and produce activity in the host body for a long time, providing experimental evidence and theoretical basis for establishing PD treatment strategies and designing new stem cell treatment drugs. The present invention has incomparable advantages over existing drugs and (or) stem cell preparations, and has broad application prospects and high commercial value in the treatment of PD.

[0008] In order to achieve the above technical effects, the present invention is implemented through the following technical scheme: a use of genetically engineered cells in the preparation of a drug for treating Parkinson's disease, characterized in that three genes encoding dopamine biosynthesis, TH (tyrosine hydroxylase), ADDC (aromatic amino acid decarboxylase), and GCH1 (GTP-cyclohydrolase 1), are introduced into the target cells for stable expression through engineering means, thereby obtaining an engineered exogenous supplement of dopamine neurotransmitters, and transplanting the cell drug into the striatum of the brain for the treatment of Parkinson's disease (PD).

[0009] Furthermore, the engineering means include one or more of adenovirus (AAV), lentivirus, transposon system, homologous recombination, and gene editing; the target cells include one or more of neural stem cells (NSC), pluripotent stem cells, mesenchymal stem cells (MSCs), etc.

[0010] Another object of the present invention is to provide a method for culturing genetically engineered cells for treating Parkinson's disease, characterized by comprising the following steps:

[0011] Step 1: Preparation of MSC cells for exogenous supplementation of dopamine neurotransmitters to treat PD;

[0012] Step 2: Obtaining MSC cells (MSC-DOPA) that can stably synthesize and secrete the neurotransmitter dopamine.

[0013] Furthermore, the specific steps in Step 1 are as follows:

[0014] Step 1.1: Obtain Wharton's jelly from the umbilical cord, aseptically shear the colloid into tissue blocks and homogenize them. Use serum-free medium to slurry the tissue blocks. Mix the homogenate by pipetting and inoculate it into a culture dish, then add serum-free medium.

[0015] Step 1.2: Culture the P0 cells, wherein the culture dish is placed in an incubator for culture; after several days of culture, the P0 cells are obtained;

[0016] Step 1.3: Cultivation of P1 generation cells, wherein the P0 generation cells are inoculated into a culture dish and placed in an incubator for culture. After the cells are confluent, the cells are digested and harvested to obtain P1 generation cells. In this way, P2, P3, P4, P5, P6... cells are obtained.

[0017] Furthermore, in Step 1.1, the colloid is cut into pieces of about 1-3 mm 3 Homogenize tissue blocks of different sizes and adjust the volume of the homogenize tissue blocks.

[0018] Furthermore, in Step 1.2, half of the medium is replaced after 6-8 days of culture; full medium is replaced after 11-14 days of culture; and cells are harvested by digestion when the cell confluence reaches 80%-90% after 15-17 days of culture to obtain P0 cells.

[0019] Furthermore, the Step 2 includes the following steps:

[0020] Step 2.1: Lentivirus preparation. Lentivirus is produced by transfecting packaging and backbone plasmids in 293T cells using the calcium phosphate / DNA coprecipitation method.

[0021] Step 2.2: Infect P0 cells with lentivirus and culture them in an incubator;

[0022] Step 2.3: Prepare cells that can stably synthesize and secrete the neurotransmitter dopamine.

[0023] Furthermore, in Step 2.1, 293T cells were cultured in Dulbecco's MEM (DMEM) containing 10% FBS; 72 hours after transfection, the supernatant containing viral particles was collected and concentrated by ultracentrifugation at 27,000 rpm for 2 hours at -4°C; and the viral particles were resuspended in DMEM.

[0024] Furthermore, the lentivirus contains three genes encoding hTH, ADDC and GCH1, and also contains genes encoding Puro and Neo.

[0025] Furthermore, in Step 2.3, the specific steps are as follows:

[0026] Step 2.3.1: 72 hours after viral infection, cells were co-treated with G418 and Puromycin to allow stable clones to grow for at least 14 days; the stable cell clones were designated MSC-DOPA-P0. After cell confluence, the cells were digested and harvested to obtain MSC-DOPA-P1 cells.

[0027] Step 2.3.2: The MSC-DOPA-P1 cells from Step 2.3.1 were seeded into a culture dish and cultured in an incubator. After the cells reached confluence, they were digested and harvested to obtain MSC-DOPA-P2 cells. MSC-DOPA-P3, -P4, -P5, ..., MSC-DOPA-P16 cells were obtained in this way.

[0028] Step 2.3.3: Screen cells;

[0029] MSC-DOPA (P0-P16) cells were tested for MSC surface antigens, and umbilical cord MSCs were screened for those with surface antigens CD90, CD73, and CD105 expression greater than 90%, and surface antigens CD45, CD34, CD19, CD14, and HLA-DR expression less than 2%. P0-P6 cells and MSC-DOPA (P1-P16) cells had the ability to transform into osteoblasts and adipocytes.

[0030] The dopamine secretion levels of cells at each generation were measured using a dopamine ELISA kit to screen cells that can stably secrete dopamine.

[0031] Furthermore, in Step 1.2, Step 1.3, Step 2.2 and Step 2.3.2, the incubator conditions are 37° C., 5% CO 2 , and saturated humidity.

[0032] The beneficial effects of the present invention are:

[0033] The present invention designs a method for culturing genetically engineered cells for treating Parkinson's disease. The MSC drug prepared by the present invention, which can exogenously supplement dopamine neurotransmitters to treat PD, has the characteristics of low cost, high activity, low immunogenicity, and significant therapeutic effect. The human MSC-DOPA produced by the present invention does not produce the drug side effect of fluctuating PD symptoms, and can survive and produce activity in the host body for a long time, providing experimental evidence and theoretical basis for establishing PD treatment strategies and designing new stem cell treatment drugs. The present invention has advantages that cannot be matched by existing drugs and (or) stem cell preparations, and has broad application prospects and high commercial value in the treatment of PD. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0035] FIG1 is a morphological diagram of the primary isolated and cultured umbilical cord mesenchymal stem cells P3 of the present invention;

[0036] FIG2 is the immunofluorescence staining result (TH+) of MSC-DOPA which can stably synthesize and secrete dopamine neurotransmitter in the present invention;

[0037] FIG3 shows the results of dopamine neurotransmitter secretion by MSC-DOPA at different generations in the present invention;

[0038] FIG4 shows the improvement results of apomorphine-induced circling behavior in PD rats before and after MSC-DOPA transplantation treatment in the present invention;

[0039] FIG5 shows the behavioral improvement results of the rotarod test in each group of PD rats before and after MSC-DOPA transplantation treatment in the present invention;

[0040] FIG6 shows the behavioral improvement results of PD monkeys before and after MSC-DOPA treatment in the present invention;

[0041] FIG7 shows the long-term (8 months) survival of MSC-DOPA transplanted into the substantia nigra of rats in each group of the present invention;

[0042] FIG8 shows the 99Tcm-TRODAT-1 SPECT brain imaging results of PD monkeys before and after MSC-DOPA treatment in the present invention;

[0043] FIG9 shows the changes in dopamine levels in the cerebrospinal fluid of each group of PD rats and PD monkeys after MSC-DOPA transplantation treatment in the present invention. DETAILED DESCRIPTION

[0044] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. 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 making any creative efforts shall fall within the scope of protection of the present invention.

[0045] Example 1

[0046] Referring to Figures 1 to 9 , the present invention provides a method for treating Parkinson's disease by supplementing dopamine neurotransmitters through genetically engineered cells, specifically comprising the following steps: 1. Preparing MSC cells capable of exogenously supplementing dopamine neurotransmitters to treat PD, and obtaining MSC cells (MSC-DOPA) that can stably synthesize and secrete the neurotransmitter dopamine. The specific steps are as follows:

[0047] 1. MSC cells that can be used to exogenously supplement dopamine neurotransmitters to treat PD are prepared by the following method:

[0048] Step 1: Obtain Wharton's jelly from the umbilical cord and cut the colloid into pieces of about 1-3 mm under sterile conditions. 3 The tissue blocks of different sizes were homogenized, and the volume of tissue blocks was fixed with homemade serum-free culture medium. The homogenate was mixed by pipetting and then inoculated into a culture dish, and serum-free culture medium was added;

[0049] Step 2: Culture the P0 cells in a 37°C, 5% CO2, and saturated humidity incubator. Change the medium halfway through the 6-8th day of culture and the full medium after the 11-14th day. Harvest the cells after the 15-17th day of culture, when the cell confluence reaches 80%-90%. This yields the P0 cells.

[0050] Step 3: Cultivation of P1 generation cells, wherein the P0 generation cells are inoculated into a culture dish and placed in an incubator under the same conditions as described in step 2 for cultivation. After the cells are confluent, the cells are digested and harvested to obtain P1 generation cells. In this way, P2, P3, P4, P5, P6 and other cells are obtained;

[0051] 2. Lentivirus preparation. Lentivirus is produced in 293T cells by transfecting packaging and backbone plasmids using the calcium phosphate / DNA co-precipitation method. 293T cells are cultured in Dulbecco's MEM (DMEM) containing 10% FBS. The supernatant containing viral particles is collected 72 hours after transfection and concentrated by ultracentrifugation at 27,000 rpm for 2 hours at -4°C. The viral particles are then resuspended in DMEM. The virus contains three genes encoding hTH, ADDC, and GCH1 for the biosynthesis of dopamine neurotransmitters; it also contains genes encoding Puromycin and G418 for screening target cells;

[0052] 3. Infect the P0 cells with the prepared lentivirus and culture them in an incubator under the same conditions as described in step 3; and prepare cells that can stably synthesize and secrete the neurotransmitter dopamine according to the following steps;

[0053] Step 1: 72 hours after viral infection, cells were co-treated with G418 and Puromycin and allowed to grow for at least 14 days. Stable cell clones were designated MSC-DOPA-P0. After cell confluence, cells were harvested and digested to obtain MSC-DOPA-P1 cells.

[0054] Step 2: The MSC-DOPA-P1 generation cells are seeded into a culture dish and placed in an incubator under the same conditions as described in step 2 for culture. After the cells are confluent, the cells are digested and harvested to obtain MSC-DOPA-P2 generation cells. MSC-DOPA-P3, -P4, -P5, etc. to MSC-DOPA-P16 cells are obtained in this way;

[0055] The P0-P6 generation cells and the MSC-DOPA (P0-P16) cells are subjected to MSC surface antigen detection to screen for umbilical cord MSCs whose surface antigens CD90, CD73, and CD105 expression are all greater than 90%, and whose surface antigens CD45, CD34, CD19, CD14, and HLA-DR expression are all less than 2%, and (or) the P0-P6 generation cells and the MSC-DOPA (P1-P16) cells are subjected to osteogenic transformation and adipogenic transformation detection to screen for umbilical cord MSCs with osteogenic and adipogenic transformation.

[0056] The MSC-DOPA (P1-P16) cells are assayed for dopamine secretion levels at each generation using a dopamine ELISA kit according to the manufacturer's method, and cells that can stably secrete dopamine are screened for PD treatment.

[0057] Example 2

[0058] 1. Preparation of a 6-OHDA-induced PD rat model. Adult Sprague Dawley rats (half male and half female) weighing 180–230 g were selected and the model was prepared and evaluated according to the following steps.

[0059] Step 1: Construction of a PD rat model. During surgery, rats were anesthetized with avertin (10 ml / kg intraperitoneally). Using bregma as the standard reference point, coordinates were selected and stereotaxically injected unilaterally with 4 μl of 6-OHDA (4 mg / ml dissolved in saline containing 0.2% ascorbic acid) directly into the right nigrostriatal pathway (at coordinates of anteroposterior [AP] = -4.4 mm, lateral [L] = -1.2 mm, and vertical [V] = -8.2 mm).

[0060] Step 2: Apomorphine-induced rotational behavioral assessment. Four weeks after stereotaxic 6-OHDA injection, rats were intraperitoneally injected with apomorphine (0.5 mg / kg). Monitoring was continued for four consecutive weeks, with each rat monitored for 30 minutes. The total number of rotations toward the side opposite to the lesion within 30 minutes was recorded. An average of more than 7 rotations per minute was considered a successful PD rat model.

[0061] 2. MSC-DOPA transplantation for PD rat model and evaluation of therapeutic effects

[0062] Step 1: Transplantation

[0063] Successful PD rat models were randomized and received MSC-DOPA transplantation. Prior to transplantation, cells were cultured in serum-free medium (SFM). MSC-DOPA were harvested by tryple digestion and, after centrifugation at 300 × g for 4 minutes, the cell pellet was resuspended in transplantation medium (Iscove's modified Dulbecco's medium, IMDM) and the viable cell concentration was adjusted to 100,000 ± 10,000 cells / ml. Using bregma as a reference, cells were injected into each site of the striatum at the following coordinates (AP = +2 mm, L = ±2.2 mm, V = -5.5 mm). Control rats received MSC or PBS instead.

[0064] Step 2: Behavioral evaluation of the therapeutic effect of MSC-DOPA transplantation

[0065] (1) Apomorphine-induced rotation behavior assessment

[0066] One week after cell transplantation, rats were intraperitoneally injected with apomorphine (0.5 mg / kg) for 12 consecutive weeks. Each rat was monitored for 30 minutes at a time, and the total number of rotations toward the side opposite to the lesion within 30 minutes was recorded. After monitoring, the data were statistically analyzed.

[0067] (2) Rotarod test

[0068] Training and testing began 14 weeks after cell transplantation. On day 1, the rotarod was trained four times over 300 seconds at a fixed speed of 4 rpm. On day 2, the rotarod was accelerated from 2 rpm to 20 rpm over 300 seconds, four times. On day 3, the rotarod was accelerated from 3 rpm to 30 rpm over 300 seconds, twice, and from 4 rpm to 40 rpm over 300 seconds, twice. Testing began on day 4, accelerating from 4 rpm to 40 rpm over 300 seconds. The time the mouse spent on the rotarod was recorded. Three to four repetitions were performed for each mouse, and the average value was used for data analysis.

[0069] Step 3: Cerebrospinal fluid (CSF) dopamine concentration measurement.

[0070] Rats were anesthetized with an intraperitoneal injection of avertin (10 ml / kg intraperitoneally). During the induction of anesthesia, the rats were kept warm in a 37°C incubator. The surgical site was scrubbed with iodine and then with 75% ethanol (repeated three times). A sagittal skin incision was made just below the occipital bone. Under a dissecting microscope, the cisterna magna dura mater appeared as a shiny, clear inverted triangle, and the medulla oblongata, major blood vessels (dorsal spinal artery), and cerebrospinal fluid spaces were visible. Cerebrospinal fluid was aspirated using a homemade capillary pipette. Cerebrospinal fluid was poured into prelabeled 0.5 ml Eppendorf tubes, immediately frozen on dry ice, and then transferred to a −80°C freezer. CSF dopamine content was determined using a dopamine ELISA kit according to the manufacturer's instructions.

[0071] Step 4: Section Staining: After euthanasia, perfuse the entire body with 4% PFA. After perfusion, remove the brain and fix it with 4% PFA overnight at 4°C. Cryosection the tissue-treated monkeys. Arrange the slides in order in a section box, label them with the tissue name and date, and store them in a -20°C refrigerator. Stain the sections with specific antibodies. Immunofluorescence staining results are captured using a fluorescence microscope.

[0072] Example 3

[0073] To construct the MPP+-induced PD monkey model, 8-15 year old macaques were selected and the model was prepared and evaluated according to the following steps.

[0074] Step 1: MPP+ induced PD monkey model

[0075] During surgery, the animals were anesthetized with ketamine hydrochloride and sodium pentobarbital. MPP+ was administered intracerebroventricularly. The bilateral modeling group received 50 μL per side, for a total of 100 μL. The unilateral group received a 100 μL injection per side, administered slowly. Injections were administered twice weekly, on Tuesday and Thursday mornings, for 15 weeks. Strict surgical aseptic procedures were adhered to throughout the surgery. A midsagittal skin incision was made, hemostasis was achieved, and muscle and connective tissue were partially removed from the opening. The skull was exposed and cleaned and disinfected with saline and 3% hydrogen peroxide. The lateral ventricle of the rhesus monkey was positioned according to predetermined coordinates. Three-dimensional positioning was used to determine the location of the drug delivery holes in the bilateral ventricles. The coordinates were 18 to 20 mm in front of the A / P zero point, 1.9 to 2.1 mm to the left and right of the midline of the brain, and 18 to 23 mm vertically. A small hole (0.82 to 0.85 mm in diameter) was drilled with a skull drill. The drug delivery tube (0.81 mm outer diameter, 0.58 mm inner diameter) was then inserted vertically using a positioning device to a depth of 18 to 22 mm below the skull surface. The ideal depth of the tube was the depth at which cerebrospinal fluid just overflowed the tube opening. In the unilateral modeling group, the tube was positioned in the left ventricle. Finally, the drug delivery tube was fixed with ceramic screws and dental cement. Within 3 days after surgery, one intramuscular injection of 1.6 million units of penicillin was given daily to prevent infection.

[0076] Step 2: Behavioral data collection

[0077] Rhesus monkeys were videotaped using a high-resolution camera (HDR-XR260, Sony) during the dosing phase, recordings were made every Wednesday and Friday afternoon. The recordings were repeated for approximately 1 hour each time after dosing, and data were collected continuously for 12 months after dosing. The behavioral videos were analyzed and behavioral scores were assessed before and after modeling using the Kurlan Scale, which uses seven key PD components: gait (0-4), tremor (0-3), body posture (0-2), overall motor function (0-3), bradykinesia (0-4), balance and coordination (0-2), and defensive reflexes (0-2). The final total score (0-20) was calculated. Video analysis was performed in a double-blind manner, with three researchers with knowledge of PD independently scoring the monkeys without knowing the monkeys' group assignments, and the average score was calculated.

[0078] Step 3: 99Tcm-TRODAT-1 SPECT brain imaging

[0079] One week after the end of drug administration, we performed 99Tcm-TRODAT-1 SPECT brain imaging in rhesus monkeys. Imaging procedures were as follows: Rhesus monkeys were anesthetized 10 minutes after intramuscular injection of atropine sulfate (0.5mg / mL, 1mL). Veterinary ketamine (0.3g / 2mL, 0.5-0.7mL) was then injected intramuscularly. Deep anesthesia was achieved through intramuscular injection of sodium pentobarbital (40mg / mL, 20mg / kg). Potassium perchlorate (10mg / kg) was then administered orally to block the choroid plexus. One hour later, 99Tcm-TRODAT-124mCi was injected through the saphenous vein. Brain imaging was performed using a Philips Precedence SPECT-CT6 system 2 hours after injection of the imaging agent. The monkeys were placed supine on an examination table. The infrared beam was aligned at the center of the face, and the head was secured for SPECT brain imaging. Acquisition conditions included a low-energy, high-resolution collimator, a magnification of 1.33, a 128×128 matrix, 360° probe rotation around the head, and acquisition of one frame every 5.6°, 60 seconds per frame, for approximately 40 minutes. Peak energy was 140 keV, and the window width was 20%. The raw images were first Butterworth filtered with a cutoff frequency of 0.4 and a steepness factor of 10. The acquired data were then reconstructed into transverse, coronal, and sagittal three-dimensional slices, with a slice thickness of 1 pixel (approximately 6 mm).

[0080] MSC-DOPA transplantation for the treatment of PD monkey models and evaluation of therapeutic effects

[0081] Step 1: Cell transplantation

[0082] All surgical procedures and nursing methods were prepared according to the reported protocols and adhered to the ethical requirements of animal research. The animal operating room was strictly disinfected and sterilized by ultraviolet for one hour before use. Rhesus monkeys were injected intramuscularly with atropine sulfate (0.5 mg / mL, 1 mL) for 10 minutes, and then anesthesia was induced by intramuscular injection of veterinary ketamine (0.3 g / 2 mL, 0.5-0.7 mL). After anesthesia, the animals were moved to the animal operating room, the hair on the top of the head was shaved, and then deep anesthesia was performed by injecting sodium pentobarbital (40 mg / mL, 20 mg / kg). The monkey head was correctly fixed on the brain stereotaxic instrument, and the scalp surface was disinfected (iodine tincture + 75% ethanol). The putamen and caudate nucleus were located according to the predetermined coordinates, with the midline of the ear as the reference, at the following coordinates (AP = 2.19 cm, ML = 1.1 cm, DV = 2.8 cm; AP = 2.52 cm, ML = 0.6 cm, DV = 2.2 cm). Then, a small hole (diameter = 0.82-0.85mm) was drilled in the skull, and 30μL of cell solution was extracted with a microinjector. The injector (OD = 0.81, ID = 0.58) was then inserted vertically using a positioning instrument. The microinjection pump was pushed in slowly and evenly at a speed of 2μl / min. For every 10μL injected, the microinjector was raised by 0.5mm to reduce the compression on the brain parenchyma. After the injection was completed, the microinjector was left in place for 3 minutes and then slowly pulled out to avoid decompression. Professional veterinarians monitored the vital signs of the surgical animals throughout the operation and kept them unconscious. Within three days after the operation, 1.6 million units of penicillin were injected intramuscularly every day to prevent infection.

[0083] Step 2: Behavioral evaluation of the therapeutic effect of MSC-DOPA transplantation

[0084] ①Behavioral data collection

[0085] Rhesus monkeys were videotaped using high-resolution video equipment during the drug administration phase, with recordings conducted once every Wednesday and Friday afternoons and continuing after drug administration. Each recording session lasted approximately 1 hour. The behavioral videos were analyzed and behavioral scores were assessed before and after modeling using the Kurlan Scale, which includes seven key PD components: gait (0-4), tremor (0-4), body posture (0-4), overall motor function (0-4), bradykinesia (0-4), balance and coordination (0-4), and defensive reflexes (0-4), with a total score (0-28). Video analysis was performed using a double-blind method, with three independent scorers averaging the scores.

[0086] ②Primate SCAN (Primate Behavior Analysis System)

[0087] Without human intervention, the system monitors the natural fine movements and behaviors of unrestrained primates in cages and automatically categorizes, counts, and analyzes them. The data intuitively reflects the rhythmicity of the animals' movements. Detectable activities include: standing upright, lowering; walking left, walking right; chewing, drinking; grooming, defecation; hanging vertically, hanging sideways; jumping, circling, twisting; crawling, lying, sitting; touching, exploring, and pausing intermittently.

[0088] ③Fingers feeding test (Fine ability test of fingers)

[0089] A turntable was mounted on a pre-prepared rectangular stand and placed in front of the monkey cage. A camera was placed on one side of the turntable to record a clear and complete video of the experiment. Eight peanuts of approximately the same size were placed in the eight holes in the second horizontal row of the turntable. The speed was set to 20 rpm, and the turntable was moved 10 cm in front of the monkey cage. The video recording began. The recording stopped after the monkey had taken all the peanuts from the turntable. The video was used to calculate the monkey's accuracy in taking the peanuts, and video analysis software was used to calculate the time it took for the monkey to remove the peanuts from the holes.

[0090] ④Visual memory experiment (Cognitive test)

[0091] A homemade rectangular box (40 mm x 40 mm, 20 mm deep) with five openings was constructed, each covered with a lid of a different color. Only the red box contained peanuts, and the position of the peanuts within each box was randomly alternating during testing. After approximately two weeks of food restriction training, when all experimental monkeys achieved nearly 100% accuracy, drug administration was initiated, and these data were collected as a control. Following drug administration, the model monkeys were tested three times per week, each round consisting of 10 repetitions. The accuracy of each of the ten repetitions was calculated, and the average of the three repetitions was used as the weekly test result. Statistical analysis of these results revealed the overall changes in cognitive function in the model monkeys throughout the drug administration period.

[0092] ⑤Emotional response intensity

[0093] In a quiet room familiar to the monkeys, a stranger who had never interacted with the monkeys stood quietly in front of the cage, 80 cm away, with their eyes looking straight ahead, maintaining the same posture for 10 minutes. The monkeys were videotaped and recorded for the number of screams, facial expressions, and movements during this time. Movement was categorized into three levels: 1 (minimal movement, such as standing and turning); 2 (intermittent movement within the cage); and 3 (constant movement).

[0094] Step 3: 99Tcm-TRODAT-1 SPECT brain imaging

[0095] One week after the end of drug administration, the model monkeys underwent 99Tcm-TRODAT-1 SPECT brain imaging. 99Tcm-TRODAT-124mCi was injected intravenously. Brain tomography was performed using a Philips Precedence SPECT-CT6 instrument 2 hours after injection of the imaging agent. The monkeys were placed supine on an examination table, with infrared light centered on the face and the head secured, for SPECT brain imaging. The raw images were first Butterworth filtered with a cutoff frequency of 0.4 and a steepness factor of 10. The acquired data were then reconstructed into three-dimensional cross-sectional, coronal, and sagittal slices, with a slice thickness of 1 pixel (approximately 6 mm). Using 99Tcm-TRODAT-1 SPECT brain dopamine transporter imaging combined with computer region of interest (ROI) technology, the ST / CB radioactivity uptake ratio was used as an indicator to semi-quantitatively analyze the DAT function of the bilateral striatum after treatment. The functionality of dopamine neurons in the striatum of the PD monkey model was compared before and after transplantation.

[0096] Step 4: CSF dopamine concentration measurement

[0097] (1) Cerebrospinal fluid collection. During the operation, ketamine hydrochloride and sodium pentobarbital were used for anesthesia. After the animal was anesthetized, the monkey was allowed to sit cross-legged with its upper body lying down. Its legs were used to fix its upper body so that it would not shake left and right. The spine was naturally protruding. The midpoint of the line connecting the ilium on the monkey's back was slightly downward and the seventh lumbar intervertebral space (between the seventh lumbar vertebra and the first sacrum) was exactly at the highest point. After the monkey was fixed, the seventh lumbar vertebra and the first sacrum were relatively prominent. The back hair was shaved off. After strict disinfection with 3% iodine tincture and 75% alcohol, the skin at the puncture site was fixed with the left thumb and index finger. The puncture needle was held in the right hand and inserted vertically. The animal could clearly feel the sense of empty space and the animal's hind limbs jumping. When there was a sense of empty space, it indicated that the needle had reached the spinal canal (subarachnoid space). At this time, the monkey's upper body was slowly uprighted, and the puncture needle core was pulled out. Transparent cerebrospinal fluid could be seen dripping out slowly. Pour the CSF into pre-labeled 1.5 ml Eppendorf tubes, freeze the tubes immediately on dry ice, and then transfer them to a -80 °C freezer.

[0098] (2) The dopamine content in CSF was determined using a dopamine ELISA kit.

[0099] Example 4

[0100] The present invention provides a method for culturing genetically engineered cells for treating Parkinson's disease. The cultured cells can be used to treat Parkinson's disease (PD). The MSC drug prepared by the present invention, which can exogenously supplement dopamine neurotransmitters to treat PD, has the characteristics of low cost, high activity, low immunogenicity, and significant therapeutic effect. The human MSC-DOPA produced by the present invention does not produce the drug side effect of fluctuating PD symptoms and can survive and produce activity in the host body for a long time, providing experimental evidence and theoretical basis for establishing PD treatment strategies and designing new stem cell treatment drugs. The present invention has advantages that are unmatched by existing drugs and / or stem cell preparations, and has broad application prospects and high commercial value in the treatment of PD.

[0101] Throughout this specification, references to terms such as "one embodiment," "example," or "specific example" indicate that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0102] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the present invention to the specific embodiments described. Obviously, many modifications and variations are possible based on the content of this specification. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for culturing genetically engineered cells for treating Parkinson's disease, characterized in that: The following steps are involved: Step 1: Preparation of MSC cells that can be used to exogenously supplement dopamine neurotransmitters to treat Parkinson's disease; Step 2: Obtaining MSC cells (MSC-DOPA) that can stably synthesize and secrete the neurotransmitter dopamine; The specific steps in Step 1 are as follows: Step 1.1: Obtain the umbilical cord, aseptically shear the colloidogen into tissue blocks and homogenize them, then use serum-free medium to make up the volume of the tissue blocks and homogenize them. After mixing by pipetting, inoculate the homogenate into a culture dish and add the culture medium. Step 1.2: Culture the P0 cells, wherein the culture dish is placed in an incubator for culture; after several days of culture, the P0 cells are obtained; Step 1.3: Cultivation of P1 generation cells, wherein the P0 generation cells are inoculated into a culture dish and placed in an incubator for cultivation. After the cells are confluent, the cells are digested and harvested to obtain P1 generation cells. This method is used to obtain P2, P3, P4, P5, P6, etc. cells; Step 2 includes the following steps: Step 2.1: Lentivirus preparation. Lentivirus is produced by transfecting packaging and backbone plasmids in 293T cells using the calcium phosphate / DNA coprecipitation method. Step 2.2: Infect P0 cells with lentivirus and culture them in an incubator; Step 2.3: Prepare cells that can stably synthesize and secrete the neurotransmitter dopamine; In Step 2.3, the specific steps are as follows: Step 2.3.1: 72 hours after viral infection, cells were co-treated with G418 and Puro, and stable clones were allowed to grow for at least 14 days; the stable cell clones were designated MSC-DOPA-P0. After cell confluence, the cells were digested and harvested to obtain MSC-DOPA-P1 cells. Step 2.3.2: Inoculate the MSC-DOPA-P1 cells from Step 2.3.1 into a culture dish and culture them in an incubator. After the cells have reached confluence, digest and harvest the cells to obtain MSC-DOPA-P2 cells. This method can be used to obtain MSC-DOPA-P3, -P4, -P5, ..., MSC-DOPA-P16 cells. Step 2.3.3: Screen the cells. MSC surface antigen detection was performed on MSC-DOPA (P0-P16) cells, and umbilical cord MSCs with surface antigens CD90, CD73, and CD105 expression greater than 90%, and surface antigens CD45, CD34, CD19, CD14, and HLA-DR expression less than 2% were selected; P0-P6 cells and MSC-DOPA (P1-P16) cells showed osteogenic and adipogenic transformation; The dopamine secretion levels of cells at each generation were measured using a dopamine ELISA kit to screen cells that can stably secrete dopamine.

2. The method for culturing genetically engineered cells for treating Parkinson's disease according to claim 1, characterized in that: The lentivirus contains three genes encoding hTH, ADDC and GCH1.

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