Preparation method of exosome derived from umbilical cord mesenchymal stem cells

Abstract

The invention belongs to the technical field of mesenchymal stem cells, and particularly relates to a preparation method of an exosome derived from umbilical cord mesenchymal stem cells. The method comprises the following steps: adding a link sequence into Nurr1 and Otx2 coding regions, connecting the Nurr1 and Otx2 coding regions with a mature sequence of miR-1246 to construct a lentiviral vector, packaging lentiviruses to obtain N-1246 lentivirus and O-1246 lentivirus, mixing the N-1246 lentivirus and O-1246 lentivirus, and jointly infecting umbilical cord mesenchymal stem cells to produce the exosome. According to the invention, Nurr1 and Otx2 coding regions are modified, so that the Nurr1 and Otx2 coding regions carry mature sequences of miR-1246 and are packaged into lentivirus infected umbilical cord mesenchymal stem cells, and exosomes containing fusion sequence mRNA are produced, so that the differentiation efficiency of neural stem cells to dopaminergic neurons is improved, and a new effective means is provided for treatment of neurodegenerative diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of mesenchymal stem cells, and particularly relates to a method for preparing exosomes derived from umbilical cord mesenchymal stem cells. Background Art

[0002] Neurodegenerative diseases, such as Parkinson's disease, are a serious threat to human health. Their primary pathological characteristic is the progressive degeneration and death of dopaminergic neurons in the midbrain. Currently, clinical treatments for neurodegenerative diseases are limited, making it difficult to fundamentally repair damaged neurons. Neural stem cells have the potential for self-renewal and multidirectional differentiation. Inducing neural stem cells to differentiate into dopaminergic neurons offers new insights into cell-based therapies for neurodegenerative diseases such as Parkinson's disease.

[0003] However, the differentiation of neural stem cells into dopaminergic neurons is strictly regulated by multiple factors. Under natural conditions, their differentiation efficiency is low, making it difficult to meet the needs of clinical treatment. In recent years, exosomes, as important mediators of intercellular communication, have garnered extensive attention in the field of cell differentiation regulation due to their ability to carry a variety of bioactive molecules (such as proteins and nucleic acids) and transmit information between cells. Human umbilical cord mesenchymal stem cells, with their abundant source, low immunogenicity, and strong proliferation capacity, are an ideal cell source for the production of exosomes.

[0004] At the molecular biological level, Nurr1 and Otx2 are key transcription factors regulating the development and differentiation of dopaminergic neurons. Mesenchymal stem cell exosomes contain a variety of RNAs, the most specific of which include miR-1246, miR-21-5p, and miR-145-5p. Not only is miR-1246 relatively abundant in mesenchymal stem cell exosomes, but studies have also shown that miR-1246 plays a crucial role in the differentiation of stem cells into neural cells. This may be achieved by targeting genes that inhibit neural differentiation and promoting the expression of genes associated with neural differentiation, thereby driving stem cell differentiation into neural cells.

[0005] Chinese patent CN 111471647A discloses a method for preparing adult stem cell exosomes and their use in treating Parkinson's disease. The method involves isolating and culturing bone marrow mesenchymal stem cells, skin epidermal stem cells, and adipose-derived stem cells in vitro. The exosomes are extracted from the cell culture supernatant by ultracentrifugation and then administered intranasally. The adult stem cell exosomes described in this patent are formed in different environments and cells, resulting in complex inclusions and a lack of druggability. Furthermore, the exosomes are unable to specifically encapsulate target proteins or mRNAs. Furthermore, these exosomes lack key transcription factors that specifically regulate the development and differentiation of neural stem cells into dopaminergic neurons. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for preparing exosomes derived from umbilical cord mesenchymal stem cells. By modifying the Nurr1 and Otx2 coding regions to carry the mature sequence of miR-1246, the lentivirus is packaged into umbilical cord mesenchymal stem cells to infect them, producing exosomes containing the fusion sequence mRNA, thereby improving the differentiation efficiency of neural stem cells into dopaminergic neurons and providing a new and effective means for the treatment of neurodegenerative diseases.

[0007] The method for preparing exosomes derived from umbilical cord mesenchymal stem cells of the present invention comprises the following steps: (1) Gene sequence modification: A link sequence was added to the 3' end of the Nurr1 coding region sequence, and the mature sequence of miR-1246 was connected to the end of the link sequence to obtain the Nurr1-link-miR-1246 fusion gene sequence. The Nurr1-link-miR-1246 fusion gene sequence is SEQ ID NO. 2; A link sequence was added to the 3' end of the Otx2 coding region sequence, and the mature sequence of miR-1246 was connected to the end of the link sequence to obtain the Otx2-link-miR-1246 fusion gene sequence. The Otx2-link-miR-1246 fusion gene sequence is SEQ ID NO. 4; (2) Lentiviral vector construction: The Nurr1-link-miR-1246 fusion gene sequence was cloned into the lentiviral expression vector to obtain pLenti-CMV-GFP-Puro-N-1246; The Otx2-link-miR-1246 fusion gene sequence was cloned into the lentiviral expression vector to obtain pLenti-CMV-GFP-Puro-O-1246; (3) Lentiviral packaging: pLenti-CMV-GFP-Puro-N-1246 was co-transfected with the lentiviral packaging plasmid into cells for lentiviral packaging to obtain N-1246 lentivirus; pLenti-CMV-GFP-Puro-O-1246 and lentiviral packaging plasmid were co-transfected into cells for lentiviral packaging to obtain O-1246 lentivirus; (4) Umbilical cord mesenchymal stem cell infection: The N-1246 lentivirus and the O-1246 lentivirus were mixed and co-infected into umbilical cord mesenchymal stem cells, which were cultured and sorted to obtain NO-1246 mesenchymal stem cells; (5) Exosome preparation: NO-1246 mesenchymal stem cells were expanded and cultured, and the cell culture supernatant was collected. The cell culture supernatant was ultracentrifuged to obtain an exosome precipitate. The exosome precipitate was resuspended in PBS to obtain an exosome solution. The exosome solution was ultrafiltered and concentrated to obtain exosomes derived from umbilical cord mesenchymal stem cells.

[0008] The Nurr1 coding region sequence in step (1) is SEQ ID NO.1.

[0009] The Otx2 coding region sequence in step (1) is SEQ ID NO.3.

[0010] The link sequence in step (1) is SEQ ID NO.5, and the mature sequence of miR-1246 is SEQ ID NO.6.

[0011] In step (1), the link sequence is used to connect the Nurr1 coding region sequence (or Otx2 coding region sequence) and the mature sequence of miR-1246 to avoid mutual interference between the two and ensure the accuracy and functionality of subsequent expression.

[0012] The lentiviral expression vector in step (2) is pLenti-CMV-GFP-Puro.

[0013] The lentiviral packaging plasmids in step (3) are psPAX2 and pMD2.G.

[0014] The cells in step (3) are 293T cells.

[0015] In step (4), the titer ratio of N-1246 lentivirus to O-1246 lentivirus is 0.8-1.2:1.

[0016] The infection time in step (4) is 12-24 hours.

[0017] In step (4), the cells expressing green fluorescent protein are sorted using flow cytometry.

[0018] The beneficial effects of the present invention are as follows: (1) Improved differentiation efficiency The present invention adds a link sequence to the Nurr1 and Otx2 coding regions and connects it to the mature sequence of miR-1246. This constructs a lentiviral vector that infects umbilical cord mesenchymal stem cells to produce exosomes, which are then co-cultured with mouse neural stem cells. Experiments have shown that, compared to traditional methods, the exosomes produced by the present invention significantly improve the differentiation efficiency of mouse neural stem cells into dopaminergic neurons after co-culture with mouse neural stem cells. Nurr1 and Otx2, as key transcription factors, can activate the expression of genes associated with dopaminergic neuron differentiation, while miR-1246 regulates gene expression by binding to target gene mRNA. The synergistic effect of these three factors provides a more favorable molecular environment for the differentiation of mouse neural stem cells into dopaminergic neurons.

[0019] (2) Good stability Lentiviral vectors can stably integrate the target gene into the mesenchymal stem cell genome, enabling the mesenchymal stem cells to consistently and stably produce exosomes containing Nurr1 and Otx2 mRNA. This stability ensures that the exosomes can continue to regulate differentiation during co-culture with mouse neural stem cells, avoiding fluctuations in differentiation efficacy caused by unstable exosome composition.

[0020] (3) Reliable source Human umbilical cord mesenchymal stem cells are abundant, relatively easy to obtain, and will not cause harm to the donor; at the same time, umbilical cord mesenchymal stem cells have low immunogenicity, which reduces the risk of immune rejection during application and improves the safety and feasibility of treatment.

[0021] (4) Broad application prospects The exosomes prepared by the present invention have important application value in the field of cell therapy for neurodegenerative diseases. By promoting the differentiation of neural stem cells into dopaminergic neurons, they can provide a new cell source and treatment strategy for the treatment of neurodegenerative diseases such as Parkinson's disease, which is expected to improve patients' symptoms and enhance their quality of life. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of N-1246.

[0023] Figure 2 This is a schematic diagram of O-1246.

[0024] Figure 3 This is the Sanger sequencing result of the pMD18-TN-1246 vector.

[0025] Figure 4 This is the Sanger sequencing result of the pMD18-TO-1246 vector.

[0026] Figure 5 This is a graph of GFP expression by N-1246 lentivirus.

[0027] Figure 6 This is a graph of GFP expression by O-1246 lentivirus.

[0028] Figure 7 This is a graph showing the cell feature identification results of umbilical cord mesenchymal stem cells. In the figure, the Count on the vertical axis represents the number of cells, and the horizontal axis represents the fluorescence intensity. A is the blank control test result without antibody labeling, B is the negative marker test result jointly labeled with five antibodies (CD34 / CD45 / CD11b / CD19 / HLA-DR), C is the positive marker test result labeled with CD105, D is the positive marker test result labeled with CD73, and E is the positive marker test result labeled with CD90.

[0029] Figure 8 This figure shows the results of sorting NO-1246 mesenchymal stem cells using flow cytometry. The area enclosed by the solid circle in the figure is NO-1246 mesenchymal stem cells expressing green fluorescent protein.

[0030] Figure 9 This is a Western blot detection result of NO-1246 mesenchymal stem cell exosomes. In the figure, lanes 1 and 2 are both Western blot detection results of NO-1246 mesenchymal stem cell exosomes.

[0031] Figure 10 This is the electrophoresis detection result of PCR products.

[0032] Figure 11 This is an immunofluorescence staining image of the dopaminergic neuron marker TH. In the figure, A is the control group, B is the experimental group, blue represents the cell nucleus stained with the nucleic acid dye DAPI, and green represents TH protein.

[0033] Figure 12 This is the result of flow cytometry analysis of dopaminergic neurons. In the figure, A, control group, B, experimental group, the part surrounded by solid circles is the proportion (%) of dopaminergic neurons generated. DETAILED DESCRIPTION

[0034] The present invention is further described below with reference to the following examples.

[0035] Example 1 (1) Obtaining Nurr1 and Otx2 coding region sequences The coding region sequences of human Nurr1 and Otx2 were obtained from the NCBI database, and the corresponding DNA fragments were obtained by chemical synthesis.

[0036] (2) Add link sequence and mature sequence of miR-1246 DNA synthesis technology was used to add a link sequence (5'-GGCGGCGGCGGCTCT-3') to the 3' end (i.e., after the stop codon) of the Nurr1 and Otx2 coding regions, respectively. The mature sequence of miR-1246 (5'-TAGTGCTTATCTGGCACTTTCC-3') was then ligated to the end of the link sequence to obtain the Nurr1-link-miR-1246 fusion gene sequence and the Otx2-link-miR-1246 fusion gene sequence, which were named N-1246 and O-1246, respectively. The schematic diagram of N-1246 is shown in the figure. Figure 1 , the schematic diagram of O-1246 is shown in Figure 2 .

[0037] (3) Cloning of N-1246 and O-1246 into pMD18-T vector The designed DNA sequences of N-1246 and O-1246 were sent to Shanghai Biotech Co., Ltd. for synthesis, and the ATG was added before the 5' end of the whole sequence. Eco RI site, added after TCC at the 3' end Hin d III site, and then cloned into the pMD18-T vector (plasmid) to obtain the pMD18-TN-1246 vector (plasmid) and the pMD18-TO-1246 vector (plasmid). After transformation into Escherichia coli DH5α, single clones were picked for expansion culture, and then the plasmids were extracted for Sanger sequencing analysis. The results are shown in Figure 3 and Figure 4 , pick out the single clone (E. coli DH5α) with correct sequencing.

[0038] (4) Construction of pLenti-CMV-GFP-Puro-N-1246 and pLenti-CMV-GFP-Puro-O-1246 lentiviral vectors a. Use respectively Eco RI and Hin pMD18-TN-1246 plasmid, pMD18-TO-1246 plasmid and pLenti-CMV-GFP-Puro plasmid were double-digested with d III. The enzyme digestion reaction system is shown in Table 1.

[0039] Table 1 Enzyme digestion reaction system

[0040] The reaction was incubated at 37°C for half an hour, and then subjected to 1.0% agarose gel electrophoresis.

[0041] b. Obtaining fragments of N-1246, O-1246 and expression vector pLenti-CMV-GFP-Puro A) Separation of double-enzyme digestion products on 1.0% agarose gel; B) Place the excised gel block into a 1.5ml EP tube and add Extraction Buffer at a volume ratio of 1:3. C) Place in a 50°C water bath for 10 minutes to completely melt the agarose, inverting and mixing every 2 minutes. Transfer the melted agarose solution into the adsorption column, centrifuge at 6000g for 1 min, pour out the liquid in the collection tube, and then place the adsorption column into the same collection tube; D) Add 500 ml of Extraction Buffer to the column and centrifuge at 12,000 g for 60 seconds. Discard the liquid in the collection tube and place the column in the same collection tube. E) Add 750 ml of Wash Buffer to the column and centrifuge at 12,000 g for 60 seconds. Discard the liquid in the collection tube and place the column in the same collection tube. F) Centrifuge again at 12,000 g for 1 min, and then place the adsorption column into a 1.5 mL centrifuge tube; G) Add 50 ml of TE buffer to the center of the column, let it stand at room temperature for 1 minute, and then centrifuge at 12,000 g for 1 minute. The resulting DNA will be stored for future use.

[0042] c. Recover the N-1246 fragment, O-1246 fragment, and pLenti-CMV-GFP-Puro vector fragment. Ligate the N-1246 fragment and O-1246 fragment with the pLenti-CMV-GFP-Puro vector fragment using DNA T4 ligase. The products were named pLenti-CMV-GFP-Puro-N-1246 and pLenti-CMV-GFP-Puro-O-1246, respectively. The ligation reaction system is shown in Table 2.

[0043] Table 2 Ligation reaction system

[0044] The ligation reaction conditions were 16°C overnight ligation, followed by transformation of E. coli DH5α.

[0045] d. Plasmids were extracted from E. coli DH5ɑ competent cells transformed with plasmids pLenti-CMV-GFP-Puro-N-1246 and pLenti-CMV-GFP-Puro-O-1246, respectively. The recombinant expression plasmids pLenti-CMV-GFP-Puro-N-1246 and pLenti-CMV-GFP-Puro-O-1246 were respectively Eco RI and Hin d III double enzyme digestion identification, 1.0% agarose gel electrophoresis detection, double enzyme digestion identification system is shown in Table 3.

[0046] Table 3 Double enzyme digestion identification system

[0047] The correctly connected recombinant expression vectors pLenti-CMV-GFP-Puro-N-1246 and pLenti-CMV-GFP-Puro-O-1246 were screened.

[0048] (5) Lentiviral packaging 293T cells in the logarithmic growth phase were digested with trypsin and 2.5 × 10 6 293T cells were re-seeded in 10cm culture dishes and cultured in a 37°C, 5% CO2 incubator until the cell confluence reached 60-70% before transfection. 32μl of the three plasmid (vector) DNA solutions in the lentiviral packaging system (pLenti-CMV-GFP-Puro-N-1246 or pLenti-CMV-GFP-Puro-O-1246 content was 20μg, psPAX2 content was 15μg, and pMD2.G content was 6μg) were gently mixed with 68μl of Lipofectamine 2000 liposomes, allowed to stand for 10 minutes, and then diluted with 1.5ml Opti-MEM, gently mixed, and incubated at room temperature for 20 minutes to form a transfection complex; the above transfection complex was then added to the cell culture dish, gently mixed, and the complete culture medium was replaced after 6 hours of culture. GFP expression was visible under an inverted fluorescence microscope 48 hours after transfection ( Figure 5 and Figure 6 );Depend on Figure 5 and Figure 6 It can be seen that green fluorescent protein expression was observed 48 hours after transfection, indicating that lentivirus was also formed synchronously; the viral supernatant was collected and centrifuged at 4°C and 3000 rpm for 5 minutes to remove cell pellets and debris; then filtered with a 0.45 μm diameter filter, and the filtered supernatant was centrifuged at 4°C and 25000 rpm for 2 hours. The viral pellet was resuspended in 500 μl PBS and dissolved at 4°C overnight to obtain N-1246 lentivirus and O-1246 lentivirus, respectively.

[0049] (6) Umbilical cord mesenchymal stem cell infection Human umbilical cord mesenchymal stem cells (huc-MSCs) were identified by flow cytometry analysis. Figure 7 ), identification indicators were observed for a blank control without antibody labeling, a negative marker co-labeled with five antibodies (CD34 / CD45 / CD11b / CD19 / HLA-DR), a positive marker labeled with CD105, a positive marker labeled with CD73, and a positive marker labeled with CD90 to determine the quality and purity of mesenchymal stem cells. Human umbilical cord mesenchymal stem cells were seeded in a 6-well plate. When the cell density reached 50-60% confluence, N-1246 lentivirus and O-1246 lentivirus were mixed at a titer ratio of 1:1. The mixed lentivirus was then co-infected with mesenchymal stem cells at an MOI of 10. Polybrene was added to a final concentration of 8 μg / ml to promote infection. After 20 hours of infection, the culture medium was replaced with DMEM containing 10% fetal bovine serum and cultured for another 50 hours. Cells expressing green fluorescent protein (GFP) were sorted by flow cytometry as target cells, thus obtaining NO-1246 mesenchymal stem cells ( Figure 8 ).

[0050] (7) Exosome preparation NO-1246 mesenchymal stem cells were washed three times with PBS, replaced with serum-free medium, and cultured for another 48 hours. The cell culture supernatant was collected and centrifuged at 4°C and 300g for 10 minutes to remove any cells that may be present in the supernatant. The supernatant was then centrifuged at 4°C and 2000g for 20 minutes to remove cell debris. The supernatant was transferred to an ultracentrifuge tube and ultracentrifuged at 4°C and 100,000g for 70 minutes. The supernatant was discarded and the precipitate was resuspended in an appropriate amount of PBS to obtain an exosome solution. The exosome solution was ultrafiltered and concentrated through an ultrafiltration tube (100kDa) to remove small molecule impurities and obtain highly pure NO-1246 mesenchymal stem cell exosomes. The expression of exosome markers CD63, CD81, and TSG101 was detected by Western blot to verify the correctness of the exosomes ( Figure 9 ).

[0051] (8) Identification of mRNA sequences in NO-1246 mesenchymal stem cell exosomes a. Total RNA Extraction The exosome total RNA extraction kit (RNAiso Plus) was purchased from Dalian TaKaRa Company. The specific steps are as follows: A) Exosomes were collected from the NO-1246 mesenchymal stem cell culture medium by centrifugation at 12,000 rpm for 5 minutes and placed in a 15 ml Corning tube. B) Add 1 mL of RNAiso Reagent, shake gently until no precipitation occurs, and transfer to a 1.5 mL EP tube; C) Place at room temperature for 5 minutes; D) Add 200 μL of chloroform, cover tightly, and shake vigorously to fully emulsify until milky white; E) Let stand at room temperature for 5 minutes; F) Centrifuge at 12000 rpm, 4°C for 15 min; G) Carefully remove the centrifuge tube. The liquid in the tube will separate into three layers. Pipette the upper layer into a new 1.5ml EP centrifuge tube, being careful not to remove the middle layer. H) Add an equal volume of isopropanol to the supernatant and mix thoroughly by gently inverting. 1) Place at room temperature for 10 minutes; J) After centrifugation at 12000 rpm and 4°C for 10 min, a white precipitate appeared at the bottom of the centrifuge tube; K) Carefully aspirate the supernatant and slowly add 1.2 ml of 75% ethanol along the tube wall, avoiding touching the pellet. Gently invert the tube to wash the tube wall. Centrifuge at 12,000 rpm and 4°C for 5 min, carefully aspirating the ethanol to ensure complete removal. M) Dry the pellet at room temperature for 5 minutes (do not centrifuge or heat dry at this step); N) Add an appropriate amount (30 μl) of RNAase-free ddH2O to dissolve the precipitate. Gently pipette the precipitate with a pipette. O) Total RNA was treated with RNase-free DNase I purchased from Dalian TaKaRa to remove trace DNA in the total RNA; The obtained total RNA was directly used for the next reverse transcription experiment or stored at -80°C.

[0052] b. cDNA Synthesis A) Measure the total RNA OD using a UV-visible spectrometer 260 With OD 280 ratio, estimating total RNA concentration and purity; B) Agarose formaldehyde denaturing gel electrophoresis to detect the quality and integrity of total RNA; C) Synthesis of cDNA: The cDNA synthesis kit was purchased from Dalian TaKaRa, and the first strand of cDNA was synthesized using Oligo dT Primer and Random 6 Mers primers; The configuration system of RT-PCR reaction solution is shown in Table 4.

[0053] Table 4 RT-PCR reaction solution configuration system

[0054] Reverse transcription conditions: 37°C for 15 min (reverse transcription reaction); 85℃5sec (inactivation reaction of reverse transcriptase); The obtained cDNA was stored at -80°C.

[0055] c. PCR primer design PCR primers were designed based on the characteristics of Nurr1 and Otx2 gene mRNA. The PCR primers are shown in Table 5.

[0056] Table 5 PCR primers

[0057] In Table 5, N-1246F is SEQ ID NO. 7, N-1246R is SEQ ID NO. 8, O-1246 F is SEQ ID NO. 9, and O-1246 R is SEQ ID NO. 10.

[0058] The PCR products were detected by electrophoresis to observe whether N-1246 mRNA and O-1246 mRNA were present in the exosomes. Figure 10 . Figure 10 The PCR results showed that two target bands were significant, indicating that N-1246 mRNA and O-1246 mRNA existed in the exosomes of NO-1246 mesenchymal stem cells.

[0059] (9) Co-culture of exosomes with mouse neural stem cells Mouse neural stem cells were transferred to new Geltrex-coated culture plates or flasks and cultured in prewarmed MEM medium. When the mouse neural stem cells reached the logarithmic growth phase, the prepared NO-1246 mesenchymal stem cell exosomes were added to the mouse neural stem cell culture system at a final concentration of 100 μg / mL as the experimental group. A control group was also established (an equal amount of PBS was added to the mouse neural stem cell culture system as the NO-1246 mesenchymal stem cell exosomes). The cells were cultured in a 37°C, 5% CO2 incubator for 7 days, with the medium replaced every 2 days and NO-1246 mesenchymal stem cell exosomes supplemented to a final concentration of 100 μg / mL. After the culture, the expression of dopaminergic neuron marker TH (tyrosine hydroxylase) was detected by immunofluorescence staining. The specific protocol was as follows: the cultured cells (experimental group and control group) were gently washed with PBS three times, 5 minutes each time; 4% paraformaldehyde (PFA) was added to fix at room temperature for 15 minutes; after fixation, the cells were washed with PBS three times, 5 minutes each time; PBS containing 0.1% Triton X-100 was added and incubated at room temperature for 10 minutes; after permeabilization, the cells were washed with PBS three times, 5 minutes each time; PBS containing 5% BSA (bovine serum albumin) was added, and 5% BSA-PBS was used as blocking solution and blocked at room temperature for 1 hour; the blocking solution was discarded and TH primary antibody diluted 1:5000 in blocking solution was added and incubated at 4°C overnight; the sections were taken out the next day and equilibrated at room temperature for 30 minutes, then washed 3 times with PBS for 10 minutes each; horseradish peroxidase (HRP)-labeled affinity-purified goat anti-mouse IgG (H+L) secondary antibody diluted 1:20000 in blocking solution was added and incubated at room temperature in the dark for 1 hour; after incubation, the sections were washed 3 times with PBS in the dark for 10 minutes each; DAPI (1 μg / mL) was added and stained at room temperature in the dark for 5 minutes; the sections were washed 2 times with PBS in the dark for 5 minutes each; the sections were sealed with anti-fluorescence quenching sealing agent and gently covered with a coverslip to avoid bubbles; the sections were observed and photographed as soon as possible under a fluorescence microscope after sealing. The results are shown in the figure. Figure 11 .Depend on Figure 11 It can be seen that the addition of NO-1246 mesenchymal stem cell exosomes significantly enhanced the expression of dopaminergic neuron marker protein TH, indicating that NO-1246 mesenchymal stem cell exosomes promote the transformation of mouse neural stem cells into dopaminergic neurons.

[0060] When mouse neural stem cells reached the logarithmic growth phase, the prepared NO-1246 mesenchymal stem cell exosomes were added to the mouse neural stem cell culture system at a final concentration of 100 μg / mL as the experimental group. A control group was also set up (PBS equal to the amount of NO-1246 mesenchymal stem cell exosomes was added to the mouse neural stem cell culture system); the cells were cultured in a 37°C, 5% CO2 incubator for 7 days, during which the culture medium was replaced every 2 days. After the culture, the cells were collected and prepared into a cell suspension, labeled with Nestin and TH antibodies, and the proportion of dopaminergic neurons was analyzed by flow cytometry ( Figure 12 ).Depend on Figure 12 It can be seen that compared with the control group, in the experimental group added with NO-1246 mesenchymal stem cell exosomes prepared by the present invention, the differentiation rate of mouse neural stem cells into dopaminergic neurons was significantly increased, proving that the exosomes prepared by the present invention can effectively regulate the differentiation of mouse neural stem cells into dopaminergic neurons.

Claims

1. A method for preparing exosomes derived from umbilical cord mesenchymal stem cells, characterized in that The steps include: (1) Gene sequence modification: A link sequence was added to the 3' end of the Nurr1 coding region sequence, and the mature sequence of miR-1246 was connected to the end of the link sequence to obtain the Nurr1-link-miR-1246 fusion gene sequence. The Nurr1-link-miR-1246 fusion gene sequence is SEQ ID NO. 2; A link sequence was added to the 3' end of the Otx2 coding region sequence, and the mature sequence of miR-1246 was connected to the end of the link sequence to obtain the Otx2-link-miR-1246 fusion gene sequence. The Otx2-link-miR-1246 fusion gene sequence is SEQ ID NO. 4; (2) Lentiviral vector construction: The Nurr1-link-miR-1246 fusion gene sequence was cloned into the lentiviral expression vector to obtain pLenti-CMV-GFP-Puro-N-1246; The Otx2-link-miR-1246 fusion gene sequence was cloned into the lentiviral expression vector to obtain pLenti-CMV-GFP-Puro-O-1246; (3) Lentiviral packaging: pLenti-CMV-GFP-Puro-N-1246 was co-transfected with the lentiviral packaging plasmid into cells for lentiviral packaging to obtain N-1246 lentivirus; pLenti-CMV-GFP-Puro-O-1246 and lentiviral packaging plasmid were co-transfected into cells for lentiviral packaging to obtain O-1246 lentivirus; (4) Umbilical cord mesenchymal stem cell infection: The N-1246 lentivirus and the O-1246 lentivirus were mixed and co-infected into umbilical cord mesenchymal stem cells, which were cultured and sorted to obtain NO-1246 mesenchymal stem cells; (5) Exosome preparation: NO-1246 mesenchymal stem cells were expanded and cultured, and the cell culture supernatant was collected. The cell culture supernatant was ultracentrifuged to obtain an exosome precipitate. The exosome precipitate was resuspended in PBS to obtain an exosome solution. The exosome solution was ultrafiltered and concentrated to obtain exosomes derived from umbilical cord mesenchymal stem cells.

2. The method for preparing exosomes derived from umbilical cord mesenchymal stem cells according to claim 1, characterized in that The Nurr1 coding region sequence in step (1) is SEQ ID NO.

1.

3. The method for preparing exosomes derived from umbilical cord mesenchymal stem cells according to claim 1, characterized in that The Otx2 coding region sequence in step (1) is SEQ ID NO.

3.

4. The method for preparing exosomes derived from umbilical cord mesenchymal stem cells according to claim 1, characterized in that The link sequence in step (1) is SEQ ID NO.5, and the mature sequence of miR-1246 is SEQ ID NO.

6.

5. The method for preparing exosomes derived from umbilical cord mesenchymal stem cells according to claim 1, characterized in that The lentiviral expression vector in step (2) is pLenti-CMV-GFP-Puro.

6. The method for preparing exosomes derived from umbilical cord mesenchymal stem cells according to claim 1, characterized in that The lentiviral packaging plasmids in step (3) are psPAX2 and pMD2.G.

7. The method for preparing exosomes derived from umbilical cord mesenchymal stem cells according to claim 1, characterized in that The cells in step (3) are 293T cells.

8. The method for preparing exosomes derived from umbilical cord mesenchymal stem cells according to claim 1, characterized in that In step (4), the titer ratio of N-1246 lentivirus to O-1246 lentivirus is 0.8-1.2:

1.

9. The method for preparing exosomes derived from umbilical cord mesenchymal stem cells according to claim 1, characterized in that The infection time in step (4) is 12-24 hours.

10. The method for preparing exosomes derived from umbilical cord mesenchymal stem cells according to claim 1, characterized in that In step (4), the cells expressing green fluorescent protein are sorted using flow cytometry.

Images