Preparation method of muc-MSC cell preparation of overexpressed exosome binding peptide-NCS1 fusion protein

Abstract

The invention belongs to the technical field of mesenchymal stem cells, and particularly relates to a preparation method of a Muc-MSC cell preparation of overexpression exosome binding peptide-NCS1 fusion protein. The method comprises the following steps: designing an exosome binding peptide sequence, constructing an exosome binding peptide-NCS1 fusion gene sequence, constructing a lentivirus for overexpressing an exosome binding peptide-NCS1 fusion protein, and constructing a muc-MSC for overexpressing the exosome binding peptide-NCS1 fusion protein. The Muc-MSC cell preparation of the overexpressed exosome binding peptide-NCS1 fusion protein, prepared by the invention, can transfer the fusion protein to target cells such as exosome, targeted neurons and glial cells, so that an endocannabinoid synthesis pathway is enhanced by the NCS1, the symptoms of the Parkinson's disease are relieved, and help is provided for future cell therapy of the Parkinson's disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of mesenchymal stem cells, and particularly relates to a method for preparing a muc-MSC cell preparation overexpressing an exosome-binding peptide-NCS1 fusion protein. Background Art

[0002] Parkinson's disease (PD), also known as "shaking palsy", is the second most common neurodegenerative disease in the world among the middle-aged and elderly. The main causes include environmental, genetic, and age factors. Its main pathological features include significant degeneration and loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain, accompanied by the formation of Lewy bodies and abnormal protein depositions such as α-synuclein aggregates in neurons. The clinical manifestations are mainly characterized by resting tremor, bradykinesia, muscle rigidity, and postural balance disorders, accompanied by non-motor symptoms such as anxiety, depression, sensory disturbances, and cognitive dysfunction. In recent years, more and more studies have suggested that PD is a multi-system disease characterized by obvious neuroinflammation and immune dysfunction.

[0003] Since the etiological mechanism of PD has not been fully elucidated, the current main treatment methods for clinical PD patients are still drug treatment and surgical treatment. Among them, drug treatment includes dopamine replacement therapy and other neurotransmitter-regulating drugs, but these can only relieve symptoms and cannot prevent the progression of the disease. Surgical treatments such as deep brain stimulation are regarded as effective options, but the long-term effects of surgical treatment are uncertain and the cost is expensive, and it cannot reverse the degeneration of dopaminergic neurons.

[0004] Therefore, various new treatment approaches, especially stem cell methods, have attracted great interest. Neural stem cells, induced pluripotent stem cells, embryonic stem cells, and mesenchymal stem cells (MSCs) have been studied for the treatment of PD. Mesenchymal stem cells (MSCs) are adult stem cells with paracrine, immunomodulatory, and multi-directional differentiation potential. In particular, human umbilical cord mesenchymal stem cells (Huc-MSCs) have multi-lineage differentiation potential, can be autotransplanted, are rich in sources and easy to obtain, and there is no ethical controversy, making them an ideal cell source for therapeutic applications. Huc-MSCs can secrete a variety of growth factors, cytokines, and chemokines, which are of great significance in key biological processes such as neuroprotection, neural differentiation, apoptosis reduction, and inflammation regulation. The exosomes (Exos) secreted by them can participate in cell-to-cell information and material exchange. At the same time, the clinical research on the treatment of diseases with Huc-MSCs is gradually increasing. However, the specific molecular mechanism of Huc-MSCs in the treatment of PD has not been elucidated. Summary of the Invention

[0005] The object of the present invention is to provide a method for preparing a muc-MSC cell preparation overexpressing an exosome-binding peptide-NCS1 fusion protein. The prepared muc-MSC cell preparation overexpressing the exosome-binding peptide-NCS1 fusion protein can transfer the fusion protein to exosomes, target target cells such as neurons and glial cells, realize that NCS1 strengthens the endogenous cannabinoid synthesis pathway, relieve the symptoms of Parkinson's disease, and provide help for the future cell therapy of Parkinson's disease.

[0006] The method for preparing a muc-MSC cell preparation overexpressing an exosome-binding peptide-NCS1 fusion protein according to the present invention comprises the following steps: (1) Design of the DNA coding sequence of the exosome-binding peptide: Design the DNA coding sequence of the exosome-binding peptide; (2) Construction of the exosome-binding peptide-NCS1 fusion gene sequence: Design and construct the exosome-binding peptide-NCS1 fusion gene sequence; (3) Construction of lentivirus overexpressing the exosome-binding peptide-NCS1 fusion protein: Clone the exosome-binding peptide-NCS1 fusion gene sequence into a vector to obtain pCDH-CMV-MCS-EF1-CopGFP-NCS1, and then co-transfect the cells with pCDH-CMV-MCS-EF1-CopGFP-NCS1 and the lentiviral packaging plasmid to obtain a lentivirus overexpressing the exosome-binding peptide-NCS1 fusion protein; (4) Construction of muc-MSC overexpressing the exosome-binding peptide-NCS1 fusion protein: Infect muc-MSC with the lentivirus overexpressing the exosome-binding peptide-NCS1 fusion protein, screen, and obtain a muc-MSC cell preparation overexpressing the exosome-binding peptide-NCS1 fusion protein after propagation and amplification.

[0007] The DNA coding sequence of the exosome-binding peptide in step (1) is SEQ ID NO.1.

[0008] The exosome-binding peptide-NCS1 fusion gene sequence in step (2) is SEQ ID NO.2.

[0009] The construction method of the exosome-binding peptide-NCS1 fusion gene sequence in step (2) is to link the DNA coding sequence of the exosome-binding peptide to the 3'-end of the NCS1 gene DNA sequence, and add a TAG stop codon to the 3'-end of the DNA coding sequence of the exosome-binding peptide to obtain the exosome-binding peptide-NCS1 fusion gene sequence.

[0010] The vector in step (3) is pCDH-CMV-MCS-EF1-CopGFP.

[0011] In step (3), the lentiviral packaging plasmids are pMD2.G, pMDLg-pRRE, and pRSV-Rev.

[0012] In step (3), the co-transfection time is 48 - 72 h.

[0013] In step (3), the cells are 293T cells.

[0014] In step (4), the infection time is 48 - 72 h.

[0015] In step (4), the screening is to use a flow cytometer to screen for muc-MSCs expressing green fluorescent protein.

[0016] The preparation method of the muc-MSC cell preparation overexpressing the exosome-binding peptide-NCS1 fusion protein according to the present invention includes the following specific steps: (1) Design of the exosome-binding peptide sequence The DNA coding sequence of the designed exosome-binding peptide is SEQ ID NO.1; (2) Construction of the exosome-binding peptide-NCS1 fusion gene sequence According to the spatial structure characteristics of the NCS1 protein, the exosome-binding peptide is linked to the C-terminus of the NCS1 protein. Correspondingly, the DNA coding sequence of the exosome-binding peptide is linked to the 3'-end of the NCS1 gene DNA sequence, and a TAG stop codon is added to the 3'-end of the DNA coding sequence of the exosome-binding peptide to obtain the exosome-binding peptide-NCS1 fusion gene sequence SEQ ID NO.2; (3) Construction of lentivirus overexpressing the exosome-binding peptide-NCS1 fusion protein The artificial synthetic exosome-binding peptide-NCS1 fusion gene sequence SEQ ID NO.2 is cloned into pCDH-CMV-MCS-EF1-CopGFP to obtain pCDH-CMV-MCS-EF1-CopGFP-NCS1. Then, pCDH-CMV-MCS-EF1-CopGFP-NCS1 is co-transfected with pMD2.G, pMDLg-pRRE, and pRSV-Rev plasmids into 293T cells. After 48 - 72 h, the lentivirus in the cell culture medium is harvested, which is the lentivirus overexpressing the exosome-binding peptide-NCS1 fusion protein; (4) Construction of muc-MSCs overexpressing the exosome-binding peptide-NCS1 fusion protein The lentivirus overexpressing the exosome-binding peptide-NCS1 fusion protein is used to infect muc-MSCs. After 48 - 72 h, muc-MSCs expressing green fluorescent protein are screened using a flow cytometer. After propagation and amplification, the muc-MSC cell preparation overexpressing the exosome-binding peptide-NCS1 fusion protein is obtained; Evaluation of the muc-MSC cell preparation overexpressing the exosome-binding peptide-NCS1 fusion protein in a Parkinson's disease mouse model A mouse model of Parkinson's disease was induced by MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). Then, the muc-MSC cell preparation overexpressing the exosome-binding peptide-NCS1 fusion protein was injected into the intramyelin sheath and the tail vein of the Parkinson's disease mice for treatment. After treatment, the therapeutic effects were evaluated from the perspectives of metabolomics and behavior.

[0017] In step (5), the MPTP induction method was intraperitoneal injection of MPTP at 28 - 32 mg / kg / d according to body weight (MPTP was dissolved in 0.9% saline before injection), and the injection was continuous for 5 - 7 days.

[0018] In the construction of the mouse model of Parkinson's disease in step (5), the experimental mice used were SPF-grade C57BL / 6 mice (male, 6 - 8 weeks old). All mice were housed in an SPF environment, freely fed with standard mouse feed, the temperature was maintained between 20 - 26 °C, and the relative humidity was maintained between 40 - 70%; the light cycle condition was 12 hours of light and 12 hours of darkness.

[0019] In step (5), the injection protocol was to perform intrathecal injection combined with tail vein injection of the muc-MSC cell preparation overexpressing the exosome-binding peptide-NCS1 fusion protein on the successfully modeled PD mouse model. The cell injection volume was 1 - 3×10 6 cells / kg; the injection frequency was once a week for 4 consecutive weeks; tail vein injection was performed in the first week, tail vein injection and intrathecal injection were performed in the second week, tail vein injection was performed in the third week, and tail vein injection and intrathecal injection were performed in the fourth week.

[0020] The evaluation protocol in step (5) included rotarod test, open field test, cerebrospinal fluid metabolomics analysis, etc.

[0021] The muc-MSC cell preparation overexpressing the exosome-binding peptide-NCS1 fusion protein prepared by the present invention can overexpress the exosome-binding peptide-NCS1 fusion protein, and can target and localize the exosome-binding peptide-NCS1 fusion protein into extracellular exosomes, and then the exosome-binding peptide-NCS1 fusion protein is secreted extracellularly together with the exosomes.

[0022] The present invention optimizes the coding sequence of the exosome-binding peptide and fuses and links it to the 3'-end of the NSC1 gene, that is, the C-terminus of the NSC1 protein, and successfully constructs the exosome-binding peptide-NCS1 fusion gene sequence; then constructs a lentivirus overexpressing the exosome-binding peptide-NCS1 fusion protein, and then infects muc-MSC cells, and screens through a flow cytometer to obtain muc-MSC cells that express the NCS1 protein and can form exosomes containing the NCS1 protein; realizes the expression of the NCS1 protein by mesenchymal stem cells that originally did not express the NCS1 protein, and at the same time realizes the encapsulation of the expressed NCS1 protein into exosomes, which is convenient for targeting glial cells and neurons in the brain environment, and lays a foundation for the treatment of Parkinson's disease and the regulation of endogenous cannabinoid production in the brain.

[0023] The present invention develops and optimizes the coding sequence of the exosome-binding peptide, establishes a new scheme for protein targeting and encapsulation into exosomes, and provides a new technology for encapsulating a target protein into exosomes by fusing the exosome-binding peptide with the target protein.

[0024] The present invention constructs muc-MSC cells expressing the exosome-binding peptide-NCS1 fusion protein, and realizes the reconstruction of the endogenous cannabinoid production signaling pathway by the NCS1 protein in the exosomes of muc-MSC cells, laying a foundation for the cell therapy of Parkinson's disease.

[0025] The present invention solves the problem of low proportion of muc-MSC cells entering the brain through the blood-brain barrier by means of combined intrathecal and intravenous injection of muc-MSC cells overexpressing the exosome-binding peptide-NCS1 fusion protein in the brain.

[0026] The present invention first designs the gene coding sequence corresponding to the exosome-binding peptide and optimizes it according to mammalian preferred codons, then artificially synthesizes the full gene sequence of the fusion protein, constructs a lentivirus overexpressing the exosome-binding peptide-NCS1 fusion protein, and then infects muc-MSC cells with the lentivirus to generate muc-MSC cells overexpressing the exosome-binding peptide-NCS1 fusion protein, and the muc-MSC cells will form exosomes containing the exosome-binding peptide-NCS1 fusion protein. The muc-MSC cell preparation overexpressing the exosome-binding peptide-NCS1 fusion protein of the present invention can strengthen the endogenous cannabinoid synthesis pathway in target cells and relieve Parkinson's symptoms.

[0027] In the present invention, a cell preparation of muc-MSCs overexpressing an exosome-binding peptide-NCS1 fusion protein is injected into the cerebrospinal fluid of a Parkinson's disease mouse model, and the limb symptoms of the model mice are effectively alleviated. By analysis, it is determined that the NCS1 protein derived from this muc-MSC cell preparation targets neurons and glial cells in the brains of Parkinson's mice, reconstructing the endogenous cannabinoid production pathway in the target cells, and the increase in endogenous cannabinoids in the cerebrospinal fluid effectively alleviates the limb symptoms of Parkinson's disease.

[0028] To achieve the continuous expression of the NCS1 protein by mesenchymal stem cells in the cerebrospinal fluid, the present invention first constructs a lentivirus overexpressing NCS1, and then infects mouse umbilical cord mesenchymal stem cells (muc-MSCs) with the lentivirus in vitro to obtain muc-MSCs overexpressing NCS1. However, the NCS1 protein is generally expressed in the cytoplasm and has no signal peptide, so it cannot be secreted outside the cell. It is found that the NCS1 protein also exists in the exosomes secreted by wild-type huc-MSCs or muc-MSCs after being intervened by human cerebrospinal fluid. However, judging from the sequence structure of the NCS1 protein, it does not belong to the main proteins secreted or entering exosomes. To increase its content in exosomes and thus target neurons or glial cells to play a role more, the present invention fuses and expresses the exosome-binding peptide "PVRASRNKRPTFLKIKKP" sequence in the NCS1 protein and the Wnt7a protein in the lentivirus. The exosome-binding peptide "PVRASRNKRPTFLKIKKP" in the Wnt7a protein can package the carried protein into exosomes in the form of a fusion protein.

[0029] To achieve the continuous administration of mesenchymal stem cells to glial cells and neurons in the brains of Parkinson's disease mice (i.e., secrete exosomes containing the NCS1 protein), the present invention first optimizes the codons of the exosome-binding peptide "PVRASRNKRPTFLKIKKP" according to mammalian preferred codons, and then constructs a lentivirus overexpressing the exosome-binding peptide-NCS1 fusion protein, transfects muc-MSCs, and obtains muc-MSCs overexpressing the exosome-binding peptide-NCS1 fusion protein. After the muc-MSC cells overexpressing the exosome-binding peptide-NCS1 fusion protein are injected into the mouse brain tissue, they can continuously express NCS1. Then, the exosome-binding peptide transports the NCS1 protein to exosomes, and then the exosomes effectively target target cells such as neurons and glial cells, realizing the reconstruction of the endogenous cannabinoid synthesis pathway by NCS1 and alleviating the symptoms of Parkinson's disease.

[0030] The beneficial effects of the present invention are as follows: Under the intervention of human cerebrospinal fluid, wild-type huc-MSCs or muc-MSCs secrete exosomes containing NCS1 protein, which target glial cells and neuronal cells to reshape the signaling pathway of endogenous cannabinoid production. The increased content of endogenous cannabinoids effectively alleviates the symptoms of Parkinson's disease and promotes the proliferation of glutamatergic neurons. However, the number of NCS1 protein exosomes formed by wild-type huc-MSCs or muc-MSCs under cerebrospinal fluid intervention is small, making it difficult to treat Parkinson's disease for a long time. In view of this, the present invention first screens exosome-binding peptide sequences that can bring target proteins into exosomes, optimizes them according to mammalian-preferred codons, fuses and links them to the C-terminus of NCS1 protein to form an exosome-binding peptide-NCS1 fusion protein sequence, and obtains muc-MSCs overexpressing the exosome-binding peptide-NCS1 fusion protein through a protocol of constructing lentivirus to infect muc-MSC cells; then constructs a Parkinson's disease model mouse, and performs cell injection treatment through the combined method of intrathecal injection and tail vein injection by puncturing the foramen magnum of the mouse occipital bone, and verifies the treatment effect from the aspects of mouse behavior and metabolomics. The muc-MSC cell preparation overexpressing the exosome-binding peptide-NCS1 fusion protein of the present invention can be used for the prevention and treatment of Parkinson's disease and other nervous system diseases, and can provide a reference for the development of similar new cells. Brief Description of the Drawings

[0031] Figure 1 It is a green fluorescent protein (GFP) expression diagram of cells containing lentivirus overexpressing the exosome-binding peptide-NCS1 fusion protein. In the figure, A is a cell diagram under the same microscope field of view and visible light, and B is a GFP green fluorescent protein diagram.

[0032] Figure 2 It is a result diagram of sorting GFP-positive muc-MSC cells by flow cytometry.

[0033] Figure 3 It is a Western blot experimental result diagram of the expression of NCS1 protein in muc-MSC-NCS1 cells, muc-MSC-NCS1 cell exosomes, MSCs cells and MSCs cell exosomes.

[0034] Figure 4 It is a result diagram of the retention time of the rotarod of Ctrl group mice and PD group mice. In the figure, represents a very significant difference between the two groups of data, and each black dot represents the retention time of a mouse.

[0035] Figure 5 It is a result diagram of the open field test of Ctrl group mice and PD group mice. In the figure, the line represents the movement trajectory of the mouse.

[0036] Figure 6It is the experimental result graph of the content of the dopaminergic neuron-specific marker TH in the substantia nigra of Ctrl group mice and PD group mice; in the graph, 1 is the staining result graph of the midbrain substantia nigra area of Ctrl group mice under 100μm, 2 is the staining result graph of the midbrain substantia nigra area of Ctrl group mice under 50μm, 3 is the staining result graph of the midbrain substantia nigra area of PD group mice under 100μm, 4 is the staining result graph of the midbrain substantia nigra area of PD group mice under 50μm, and the dopaminergic neurons marked in the area circled by the dotted line are shown in brown.

[0037] Figure 7 It is a schematic diagram of the treatment plan for mice by injecting muc-MSC-NCS1 cells.

[0038] Figure 8 It is the result graph of the non-targeted metabolomics analysis of the cerebrospinal fluid of MSCPD group mice.

[0039] Figure 9 It is the result graph of the retention time of the rotarod of PD group mice and MSCPD group mice. Represents a very significant difference between the two groups of data, and each black dot represents the retention time of a mouse.

[0040] Figure 10 It is the result graph of the open field test of PD group mice and MSCPD group mice. In the graph, the lines represent the movement trajectories of the mice. Detailed implementation manners

[0041] The present invention will be further described below in conjunction with embodiments.

[0042] Embodiment 1 (1) Design of exosome-binding peptide sequence Translate the exosome-binding peptide "PVRASRNKRPTFLKIKKP" into the corresponding DNA codons, and then optimize it according to mammalian preferred codons to obtain the DNA coding sequence SEQ ID NO.1 of the exosome-binding peptide; (2) Construction of exosome-binding peptide-NCS1 fusion gene sequence Analyze the spatial structure characteristics of the NCS1 protein. On the basis of ensuring that the exosome-binding peptide does not affect the spatial conformation of the NCS1 protein, link the coding sequence of SEQ ID NO.1 to the 3'-end of the NCS1 gene DNA sequence, and add a TAG stop codon to the 3'-end of the SEQ ID NO.1 sequence, thereby obtaining the exosome-binding peptide-NCS1 fusion gene sequence SEQ ID NO.2; (3) Construction of lentivirus overexpressing exosome-binding peptide-NCS1 fusion protein: The exosome-binding peptide-NCS1 fusion gene sequence SEQ ID NO.2 was synthesized by artificial chemical synthesis method, and then cloned into the pCDH-CMV-MCS-EF1-CopGFP plasmid to obtain the pCDH-CMV-MCS-EF1-CopGFP-NCS1 plasmid; The 293T cells in the logarithmic growth phase were digested with trypsin, and 2.5×10 6 cells were re-seeded in a 10 cm culture dish and cultured in an incubator at 37 °C and 5% CO 2 until the cell confluence reached 60-70% before transfection; the lentiviral packaging system included pCDH-CMV-MCS-EF1-CopGFP-NCS1, pMD2.G, pMDLg-pRRE and pRSV-Rev. Take 10 μg of pCDH-CMV-MCS-EF1-CopGFP-NCS1 plasmid, 3 μg of pMD2.G plasmid, 7 μg of pMDLg-pRRE plasmid and 5 μg of pRSV-Rev plasmid and place them in the same 1.5 ml EP tube. Add Opti-MEM cell culture medium to a volume of 250 μl and mix well, named tube 1; take another EP tube, add 1.5 ml of Opti-MEM cell culture medium and 60 μl of Lipofectamine 2000 liposome, mix well and name it tube 2; then gently add 250 μl of the mixture from tube 2 to tube 1 and mix well, incubate at room temperature for 20 min to form a transfection complex; then add the above transfection complex to the cell culture dish, mix gently, change the complete medium after culturing for 6 h, and GFP expression can be seen under an inverted fluorescence microscope 48 h after transfection ( Figure 1 ); collect the virus supernatant, centrifuge at 3000 r / min at 4 °C for 5 min to remove cell pellets and debris; then filter through a 0.45 μm filter, and centrifuge the filtered supernatant at 25000 r / min at 4 °C for 2 h. Resuspend the virus pellet with 500 μl of PBS and dissolve overnight at 4 °C to obtain the lentivirus overexpressing the exosome-binding peptide-NCS1 fusion protein; (4)Construction of muc-MSC overexpressing exosome-binding peptide-NCS1 fusion protein muc-MSC was seeded in a 24-well plate at a density of 2.0×10 5 cells / well, and the experiment started 12 h later; 300 μl of the lentivirus overexpressing the exosome-binding peptide-NCS1 fusion protein was added to each well for infection, and polybrene was added to a final concentration of 5 μg / ml to increase the infection efficiency; the next day, the virus-containing medium was removed and replaced with serum-free complete medium. 48 h after infection, GFP-positive muc-MSC cells were sorted by flow cytometry, and the cells with strong GFP fluorescence intensity were selected as the target cells ( Figure 2), that is, muc-MSCs overexpressing the exosome-binding peptide-NCS1 fusion protein were obtained, named muc-MSC-NCS1 cells; after the muc-MSC-NCS1 cells were propagated and amplified, a muc-MSC cell preparation overexpressing the exosome-binding peptide-NCS1 fusion protein was obtained.

[0043] Analysis and detection: A. Analysis of NCS1 protein expression in muc-MSC-NCS1 cell exosomes Separate muc-MSC-NCS1 cells and muc-MSC-NCS1 cell exosomes, as well as MSCs cells and MSCs cell exosomes. After treatment with protein lysis buffer and centrifugation, take the supernatant. After quantifying the protein by the BCA method, the loading amount is 10 µl. The electrophoresis starts at 80 v for 30 minutes and then 120 v until the end. Transfer the membrane at 200 mA for 50 minutes. After blocking with 5% skim milk for 1 hour, cut the membrane. Add the primary antibodies NCS1 and CD63 and incubate overnight at 4°C (the exosome-specific protein CD63 is used as the internal reference protein). Wash with TBST and then incubate with the secondary antibody (HRP-labeled goat anti-mouse IgG) at room temperature for 1.5 hours. Finally, develop the color with the ECL developer. The results are as Figure 3 shown: The NCS1 protein was significantly expressed in muc-MSC-NCS1 cells and muc-MSC-NCS1 cell exosomes, but not expressed in MSCs cells and MSCs cell exosomes, indicating that muc-MSC-NCS1 cells can express the NCS1 protein and can coat the protein into exosomes.

[0044] B. Construction of a Parkinson's disease mouse model and behavioral detection Experimental mice were SPF-grade C57BL / 6 mice (male, 6 - 8 weeks old). Among them, there were 10 mice in the control group (Ctrl) and 10 mice in the Parkinson's disease model group (PD). The modeling method adopted the protocol for MPTP model PD mice (Q.S. Zhang, Y. Heng, Z. Mou, J.Y. Huang, Y.H. Yuan, N.H. Chen, Reassessment of subacute MPTP-treated mice as animal model of Parkinson's disease, Acta pharmacologica Sinica, 38(2017) 1317 - 1328). On this basis, the modeling protocol was slightly adjusted: All mice had an adaptation period of 1 week before the experiment started. All mice were housed in an SPF environment, fed ad libitum with standard mouse feed, the temperature was maintained between 20 - 26 °C, and the relative humidity was maintained between 40 - 70%; the light cycle condition was 12 hours of light and 12 hours of darkness. After the adaptation period ended, the 10 mice in the PD group were weighed, and MPTP 30 mg / kg / d (dissolved in 0.9% saline) was intraperitoneally injected according to their body weight for 5 consecutive days. After the injection ended, behavioral tests were conducted on all mice, including the rotarod test and the open field test.

[0045] The rotarod test evaluated the motor coordination of mice. First, the mice were trained at 5 - 10 r / min for 300 s. After more than 3 rounds of training, the formal experiment was carried out and the time from when the mice got on the rotarod to when they fell off was recorded. The results showed that, compared with the mice in the Ctrl group, the retention time of the PD group mice on the rotarod decreased significantly ( Figure 4 ). The open field test was conducted in an empty box (50 cm × 50 cm × 50 cm) that recorded the mouse trajectory in real time. Before the test, the mice were pre-adapted for 5 minutes. The next day, for the formal experiment, the mice were placed in the center and a 5-min video was recorded. Each time, the device was cleaned with medical alcohol and water. The experimental results showed ( Figure 5In the open field test, both the movement distance and the number of wall touches of the PD group mice showed significant decreases. Whole brains were taken from the euthanized Ctrl group and PD group mice for tyrosine hydroxylase (TH) immunohistochemical identification. First, the left atrium of the mice was perfused with 15 ml of PBS, and then the brains were taken. They were fixed with 4% paraformaldehyde for more than 24 h, then dehydrated, embedded in paraffin blocks and sectioned to the substantia nigra region of the brain (section thickness 4 μm). After that, the paraffin sections were baked in an oven, dewaxed with xylene, soaked in ethanol of different concentrations, treated with hydrogen peroxide, antigen repaired, blocked, incubated with TH antibody, washed, incubated with secondary antibody, washed, developed with DAB under the microscope, and finally the midbrain SN was photographed under an upright optical microscope. The TH immunohistochemical staining of the mice would stain the dopaminergic neurons in the substantia nigra region of the mice brown, and thus judge the change in the number of neurons in the substantia nigra region. The identification results of the present invention showed that the content (brown area) of the dopaminergic neuron specific marker TH in the substantia nigra of PD mice also showed a significant decrease ( Figure 6 ). Therefore, considering the results of the behavioral detection of the two groups of mice and the TH staining of the mouse brain, the constructed MPTP model PD mice conformed to the relevant pathological manifestations of PD.

[0046] C. muc-MSC-NCS1 cell injection treatment of PD mice The successfully modeled PD mouse models were randomly divided into a Parkinson's disease model (PD) group and a muc-MSC-NCS1 cell treatment (MSCPD) group. The MSCPD group was treated with muc-MSC-NCS1 cells by the combined method of intrathecal injection and tail vein injection using the method of puncturing the foramen magnum of the mouse brain ( Figure 7 ), and the cell injection volume was 1×10 6 cells / kg; the injection frequency was once a week for 4 consecutive weeks. In the first week, cerebrospinal fluid was taken after puncturing the foramen magnum of the mouse brain, 5 μl was taken from each mouse, and then tail vein injection was carried out according to the standard of 1×10 6 cells / kg. In the second week, after tail vein injection according to the standard of 1×10 6 cells / kg, intrathecal injection treatment was carried out according to the standard of 1×10 6 cells / kg. In the third week, tail vein injection was carried out according to the standard of 1×10 6 cells / kg. In the fourth week, after tail vein injection according to the standard of 1×10 6 cells / kg, intrathecal injection treatment was carried out again according to the standard of 1×10 6Intrathecal injection treatment was performed at the standard of cells / kg. Mouse cerebrospinal fluid (CSF) was collected before treatment and at the 8th week after treatment. Method for collecting mouse CSF: Collection was performed by puncturing through the foramen magnum. After anesthetizing and fixing the mouse, the foramen magnum area was fully exposed, and a micro syringe was carefully inserted for slow aspiration of CSF. After collecting the mouse CSF, non-targeted metabolomics analysis was carried out, and subsequent behavioral tests and evaluations were also performed.

[0047] D. CSF metabolomics analysis The CSF collected in C was subjected to non-targeted metabolomics analysis. Using the PD group as a control, the changes in endogenous cannabinoids in the CSF of mice after muc-MSC-NCS1 cell injection treatment were observed. The metabolomics analysis was performed by Suzhou Panomic Biotech Co., Ltd. using liquid chromatography-tandem mass spectrometry for non-targeted analysis. After data preprocessing and annotation, multivariate statistical analysis was carried out. The results of the changes in differential metabolites showed that endogenous cannabinoids, guanosine, dihydrouracil, etc. were significantly up-regulated ( Figure 8 ), indicating that the exosomes containing NSC1 protein produced by muc-MSC-NCS1 cells effectively promoted the synthesis of exogenous cannabinoids in glial cells or neurons.

[0048] E. Behavioral tests for the treatment effect of muc-MSC-NCS1 cell injection in PD mice Rotarod tests and open field tests were performed on the mice in the Parkinson's disease model (PD) group and the muc-MSC-NCS1 cell treatment (MSCPD) group in C. The experimental results of the rotarod test showed ( Figure 9 ), that the time the mice in the MSCPD group stayed on the rotarod after treatment was significantly increased compared to the PD group; the results of the open field test showed ( Figure 10 ), that the movement distance and the number of wall touches of the mice in the MSCPD group were both significantly increased compared to the PD group in the open field test.

[0049] In summary, the muc-MSC-NCS1 cells constructed in the present invention can not only effectively express the NCS1 protein, but also package the NCS1 protein into exosomes to achieve delivery to target cells (glial cells and neurons). NCS1 can increase the content of endogenous cannabinoids in the brain and fully relieve the symptoms of Parkinson's disease. Therefore, the muc-MSC cell preparation overexpressing the exosome-binding peptide-NCS1 fusion protein prepared in the present invention is a novel immune cell preparation and can be used for the prevention and treatment of Parkinson's disease.

Claims

1. A method for preparing a muc-MSC cell preparation overexpressing an exosome-binding peptide-NCS1 fusion protein, characterized in that The steps include: (1) Exosome-binding peptide sequence design: Design the DNA coding sequence of the exosome-binding peptide; (2) Construction of exosome-binding peptide-NCS1 fusion gene sequence: Design and construct the exosome-binding peptide-NCS1 fusion gene sequence; (3) Construction of lentivirus overexpressing exosome-binding peptide-NCS1 fusion protein: The exosome-binding peptide-NCS1 fusion gene sequence was cloned into the vector to obtain pCDH-CMV-MCS-EF1-CopGFP-NCS1, and then pCDH-CMV-MCS-EF1-CopGFP-NCS1 was co-transfected with the lentivirus packaging plasmid into the cells to obtain a lentivirus overexpressing the exosome-binding peptide-NCS1 fusion protein; (4) Construction of muc-MSCs overexpressing exosome-binding peptide-NCS1 fusion protein: Infect muc-MSCs with lentivirus overexpressing exosome-binding peptide-NCS1 fusion protein, and obtain muc-MSC cell preparations overexpressing exosome-binding peptide-NCS1 fusion protein after screening, propagation and amplification; The DNA coding sequence of the exosome-binding peptide in step (1) is SEQ ID NO.

1.

2. The method for preparing a muc-MSC cell preparation overexpressing an exosome-binding peptide-NCS1 fusion protein according to claim 1, characterized in that The exosome binding peptide-NCS1 fusion gene sequence in step (2) is SEQ ID NO.

2.

3. The method for preparing a muc-MSC cell preparation overexpressing an exosome-binding peptide-NCS1 fusion protein according to claim 1, characterized in that In step (2), the exosome binding peptide-NCS1 fusion gene sequence is constructed by linking the DNA coding sequence of the exosome binding peptide to the 3'-end of the NCS1 gene DNA sequence, and adding a TAG stop codon to the 3'-end of the DNA coding sequence of the exosome binding peptide to obtain the exosome binding peptide-NCS1 fusion gene sequence.

4. The method for preparing a muc-MSC cell preparation overexpressing an exosome-binding peptide-NCS1 fusion protein according to claim 1, characterized in that The vector in step (3) is pCDH-CMV-MCS-EF1-CopGFP.

5. The method for preparing a muc-MSC cell preparation overexpressing an exosome-binding peptide-NCS1 fusion protein according to claim 1, characterized in that In step (3), the lentiviral packaging plasmids are pMD2.G, pMDLg-pRRE and pRSV-Rev.

6. The method for preparing a muc-MSC cell preparation overexpressing an exosome-binding peptide-NCS1 fusion protein according to claim 1, characterized in that The total transfection time in step (3) is 48-72h.

7. The method for preparing a muc-MSC cell preparation overexpressing an exosome-binding peptide-NCS1 fusion protein according to claim 1, characterized in that The cells in step (3) are 293T cells.

8. The method for preparing a muc-MSC cell preparation overexpressing an exosome-binding peptide-NCS1 fusion protein according to claim 1, characterized in that The infection time in step (4) is 48-72h.

9. The method for preparing a muc-MSC cell preparation overexpressing an exosome-binding peptide-NCS1 fusion protein according to claim 1, characterized in that In step (4), the screening is to screen muc-MSCs expressing green fluorescent protein using flow cytometry.

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