Three-dimensional contact co-culture system for bone marrow mesenchymal stem cells and neurons and construction method thereof

Abstract

The invention belongs to the technical field of cell biology and tissue engineering, and particularly relates to a bone marrow mesenchymal stem cell and neuron three-dimensional contact co-culture system and a construction method thereof. According to the method, the bone marrow mesenchymal stem cells and the neurons are inoculated on the polylactic acid-glycolic acid porous cell scaffold for co-culture, so that the three-dimensional contact state of the bone marrow mesenchymal stem cells and the neurons during intracerebral cell transplantation can be better simulated, the contact area of the bone marrow mesenchymal stem cells and the neurons is increased, an in-vitro intercellular contact model is established, and the in-vitro intercellular contact model is established. The device is used for researching the mechanical acting force between contact cells, intercellular communication and the like. Besides, the bone marrow mesenchymal stem cells are marked by green fluorescent protein, so that the bone marrow mesenchymal stem cells can be distinguished from neurons under a fluorescence microscope, and the bone marrow mesenchymal stem cells can be easily separated from mixed cells of the bone marrow mesenchymal stem cells and the neurons, so that a foundation is laid for subsequent research.

Description

Technical Field

[0001] The invention belongs to the technical field of cell biology and tissue engineering, and specifically relates to a three-dimensional contact co-culture system of bone marrow mesenchymal stem cells and neurons and a construction method thereof. Background Art

[0002] In recent years, both basic research and clinical trials have shown that bone marrow mesenchymal stem cells are one of the ideal seed cells for transplantation to treat central nervous system diseases, but the biological characteristics of bone marrow mesenchymal stem cells in the brain are not yet fully understood. As far as current research is concerned, on the one hand, the beneficial mechanisms of bone marrow mesenchymal stem cells on central nervous system diseases mainly include cell replacement, inhibition of neuroinflammation, promotion of angiogenesis, and secretion of cytokines; on the other hand, the neural tissue microenvironment also acts on bone marrow mesenchymal stem cells through some cytokines, growth factors, etc. However, there are few studies on the effect of neuronal contact, a factor that cannot be ignored in the neural tissue microenvironment, on the biological characteristics of bone marrow mesenchymal stem cells.

[0003] The Chinese patent application number 201510504338.5 discloses a three-dimensional co-culture induction method for mesenchymal stem cells, which includes encapsulating mesenchymal stem cells and magnetic oxides in gel ball A; encapsulating target cell sources in gel ball B; co-culturing the mesenchymal stem cells in gel ball A and the target cell sources in gel ball B with an inducer until the mesenchymal stem cells are transformed into target cells, and then placing gel ball A and gel ball B in a magnetic field for separation. The patent establishes a method for co-culturing mesenchymal stem cells and target cells and separating the two cells. However, this method cannot truly simulate the contact state between mesenchymal stem cells and host cells during transplantation, and ignores the mechanical force and intercellular communication between contacting cells, as well as the migration characteristics of mesenchymal stem cells.

[0004] Cells can not only influence each other by secreting cytokines, but also respond to mechanical forces and intercellular communication between neighboring cells. Ordinary single-layer cell culture and double-layer cell culture are difficult to simulate the three-dimensional contact state between bone marrow mesenchymal stem cells and neurons during brain transplantation. Summary of the invention

[0005] The purpose of the present invention is to utilize tissue engineering and cell biology techniques to inoculate mixed bone marrow mesenchymal stem cells labeled with green fluorescent protein and neurons on a polylactic acid-glycolic acid porous cell scaffold for co-culture, thereby achieving three-dimensional contact between neurons and bone marrow mesenchymal stem cells and simulating the construction of an in vitro cell transplantation model. The bone marrow mesenchymal stem cells and neurons can also be separated by utilizing green fluorescent protein labeling, based on which the contact between the two cells is observed, thereby providing technical methods and experimental tools for numerous subsequent studies.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] First, the present invention provides a method for constructing a three-dimensional contact co-culture system of bone marrow mesenchymal stem cells and neurons, comprising the following steps:

[0008] S1, dissolving lactic acid and glycolic acid in an organic solvent, mixing with a porogen, and then placing in a mold for forming, demoulding, removing the sodium chloride porogen, and drying to obtain a polylactic acid-glycolic acid porous cell scaffold, cutting the polylactic acid-glycolic acid porous cell scaffold into a specific size, and then disinfecting, cleaning, and soaking in polylysine in sequence, placing the treated polylactic acid-glycolic acid porous cell scaffold in a first culture plate, drying under sterile conditions, and then adding α-MEM into the wells to soak overnight, and transferring the treated polylactic acid-glycolic acid porous cell scaffold to a second culture plate for drying;

[0009] S2, preparing a cell suspension of bone marrow mesenchymal stem cells labeled with green fluorescent protein;

[0010] S3. Preparation of cell suspension of primary neurons;

[0011] S4. Mix the cell suspension of bone marrow mesenchymal stem cells labeled with green fluorescent protein with the cell suspension of neurons, and then inject them into the polylactic acid-glycolic acid porous cell scaffold in the second culture plate for culture at multiple points. After 3-4 hours, change the medium, discard the culture medium and non-adherent cells, add a neuron-specific culture medium containing cytarabine and continue to culture for 6-7 days to obtain a three-dimensional contact co-culture system of bone marrow mesenchymal stem cells and neurons.

[0012] Furthermore, in S1, the molar ratio of lactic acid to glycolic acid is 80:20.

[0013] Furthermore, in S1, the organic solvent is dichloromethane, and the mass volume ratio of lactic acid and glycolic acid to dichloromethane is 1g:6-10ml.

[0014] Furthermore, in S1, the porogen is sodium chloride particles with a particle size of 180 to 250 μm.

[0015] Furthermore, in S1, the mass ratio of lactic acid and glycolic acid to the porogen is 1:15 to 1:22.

[0016] Further, in S2, preparing a cell suspension of bone marrow mesenchymal stem cells labeled with green fluorescent protein comprises the following steps:

[0017] A1. Isolate the bilateral tibiae and femurs of 1-month-old rats, and flush the bone marrow cavity with phosphate buffer to obtain a cell suspension;

[0018] A2, adding the cell suspension described in A1 onto the rat lymphocyte separation solution, and collecting the interface cell layer by centrifugation;

[0019] A3, resuspending the cells of the interface cell layer described in A2 in α-MEM medium containing 10% fetal bovine serum, inoculating them in a culture flask and culturing them for 2 hours;

[0020] A4: Replace the entire volume of medium to remove non-adherent cells and impurities in A3, and replace the medium every 2 to 3 days thereafter;

[0021] When the cells obtained from A5 and A4 reached about 80% confluence, the cells were digested with trypsin, then centrifuged and resuspended in α-MEM medium containing 10% fetal bovine serum for further subculture;

[0022] When the passaged A6 and A5 cells reached 60%-70% confluence, recombinant lentivirus carrying the green fluorescent protein gene was added and incubated for 48 hours;

[0023] The cells cultured in A7 and A6 were digested with trypsin digestion solution, then centrifuged to remove the supernatant, resuspended in fresh culture medium, and continued to be cultured, and the fluorescence expression was observed in 24-48 hours;

[0024] After digestion, the cultured cells of A8 and A7 were resuspended and the cell concentration was adjusted to 10 cells / mL. 100 μL of cell suspension was added to each well of the 96-well plate. The wells expressing GFP and single cells were observed and selected under an inverted fluorescence microscope for labeling and expansion of culture.

[0025] Further, in S3, preparing a cell suspension of primary neurons comprises the following steps:

[0026] B1. Isolate the cerebral cortex of newborn rats within 24 hours of birth and cut it into pieces. Add digestive enzyme solution and place it in a 37°C incubator for digestion. During the digestion, pipette and perfuse 1-2 times.

[0027] B2, adding DMEM culture medium containing 10% fetal bovine serum to the brain tissue of B1 to terminate digestion and prepare a suspension, and dropping it into a cell sieve with a pore size of 100 μm to obtain a first cell suspension;

[0028] B3. After centrifuging the first cell suspension of B2, remove the supernatant, resuspend the precipitate in α-MEM culture medium containing 10% fetal bovine serum, and let it stand for 5-10 minutes to remove fibroblasts to obtain a second cell suspension, i.e., the cell suspension of primary neurons.

[0029] Furthermore, in S4, the ratio of green fluorescent protein-labeled bone marrow mesenchymal stem cells to neurons was 1:2-4.

[0030] Secondly, the present invention provides a three-dimensional contact co-culture system of bone marrow mesenchymal stem cells and neurons constructed by the above construction method.

[0031] Beneficial effects:

[0032] (1) The present invention constructs a three-dimensional contact co-culture system of GFP-labeled bone marrow mesenchymal stem cells and neurons through bioengineering and cell biology methods, which can more realistically simulate the three-dimensional contact state of bone marrow mesenchymal stem cells and neurons during brain transplantation, the mechanical force and intercellular communication between contacting cells, and the migration characteristics of mesenchymal stem cells, and can be used for in vitro cell experimental research to make the research results more accurate.

[0033] (2) The prepared polylactic acid-glycolic acid porous cell scaffold has high porosity, good pore connectivity, and appropriate pore size, which is conducive to cell migration and contact.

[0034] (3) By selecting the appropriate inoculation amount of GFP-labeled bone marrow mesenchymal stem cells and neurons on the polylactic-co-glycolic acid porous cell scaffold, the cell's nutrient acquisition and metabolic waste discharge are ensured, thereby promoting cell metabolism.

[0035] (4) By selecting the appropriate seeding ratio of GFP-labeled bone marrow mesenchymal stem cells and neurons on the polylactic acid-glycolic acid porous cell scaffold, the contact area between bone marrow mesenchymal stem cells and neurons is increased, thereby better simulating the contact condition between bone marrow mesenchymal stem cells and neurons transplanted into the brain.

[0036] (5) Bone marrow mesenchymal stem cells are labeled with GFP to facilitate their distinction from neurons under a fluorescence microscope; the two mixed cells can also be separated using flow cytometry to facilitate subsequent research. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is the second generation of bone marrow mesenchymal stem cells.

[0038] Figure 2 Bone marrow mesenchymal stem cells labeled with GFP.

[0039] Figure 3 These are primary cultured cerebral cortical neurons.

[0040] Figure 4 This is a scanning electron image of the surface, interior and interstitial connectivity of the poly(lactic-co-glycolic acid) porous cell scaffold.

[0041] Figure 5 Scanning electron microscopy shows the growth of bone marrow mesenchymal stem cells and neurons seeded on poly(lactic-co-glycolic acid) porous cell scaffolds.

[0042] Figure 6 Immunofluorescence staining shows neural differentiation of bone marrow mesenchymal stem cells (A), and the statistical graph of the proportion of bone marrow mesenchymal stem cells expressing NeuN (B). Green is bone marrow mesenchymal stem cells, red is NeuN immunofluorescence staining, and blue is DAPI staining. DLC: double-layer co-culture group; CC: contact co-culture group.

[0043] Figure 7 Are the expression levels of Notch-1 mRNA and protein, A is the statistical graph of Notch-1 mRNA expression level, B is the Western Blot graph of Notch-1 protein, and C is the statistical graph of Notch-1 protein expression level; DLC: double-layer co-culture group; CC: contact co-culture group. DETAILED DESCRIPTION

[0044] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0045] Example 1

[0046] The three-dimensional contact co-culture system of bone marrow mesenchymal stem cells and neurons provided in this embodiment is constructed as follows:

[0047] 1. Preparation of polylactic acid-glycolic acid porous cell scaffold:

[0048] 1.1. Lactic acid and glycolic acid are dissolved in dichloromethane at a molar ratio of 80:20 (the mass volume ratio of the two to dichloromethane is 1g:9ml), and then screened sodium chloride particles with a diameter of 180-250μm are added. Among them, lactic acid / glycolic acid and sodium chloride are mixed at a mass ratio of 1:20.

[0049] 1.2. Stir the above mixture evenly and pour it into the prepared mold (50mm×50mm×3mm), and shape it at room temperature and normal pressure. Take out the semi-finished product and soak it in double distilled water. Change the double distilled water several times to completely release the sodium chloride particles, and then place it in a dryer for vacuum drying to obtain a polylactic acid-glycolic acid porous cell scaffold and store it in a vacuum sealed plastic bag to prevent contamination.

[0050] 1.3. Before use in the experiment, the polylactic acid-glycolic acid porous cell scaffold was cut into small pieces of 10 mm × 10 mm × 3 mm in size, immersed in 75% alcohol for 2 hours for disinfection, then washed with sterile double distilled water for 3 times and then immersed in poly-lysine for 2 minutes.

[0051] 1.4. Place the polylactic acid-glycolic acid porous cell scaffold treated as above into a 24-well culture plate and place it on a sterile workbench to dry. Then add α-MEM culture medium into the wells and soak it overnight. Absorb the excess culture medium and dry it on a sterile workbench for later use.

[0052] 2. Cultivation of bone marrow mesenchymal stem cells and green fluorescent protein labeling

[0053] 2.1. SD rats weighing about 100 g were killed by cervical dislocation and immersed in 75% ethanol for 3 minutes for disinfection. The rats were fixed in a supine position, the skin of both hind limbs was cut open, and the bilateral tibiae and femurs were separated and placed in a culture dish containing PBS.

[0054] 2.2. Separate the muscle tissue from the tibia and femur, rinse twice with PBS, and transfer to a new culture dish.

[0055] 2.3. After cutting off the epiphysis at both ends of the bone, use a sterile syringe to draw 5 ml of α-MEM culture medium to flush the bone marrow cavity. Use a pipette to blow the bone marrow flushing fluid 3-5 times and transfer it into a centrifuge tube. Centrifuge at 1000 rpm for 5 minutes, remove the fat layer, and resuspend the cells with PBS solution.

[0056] 2.4. Slowly add the above bone marrow cell suspension into a centrifuge tube pre-filled with 5 ml of rat lymphocyte separation solution (P6030-200 ml / Kit), centrifuge at 800 rpm for 20 minutes, carefully pipette the middle interface layer cells into the centrifuge tube, add 3 ml of PBS solution, and centrifuge and wash once.

[0057] 2.5. Resuspend the cells in α-MEM medium containing 10% fetal bovine serum, inoculate in a culture flask, and place in a 37°C, 5% carbon dioxide incubator.

[0058] 2.6. After culturing the cells for 2 hours, replace the medium completely to remove non-adherent cells and impurities. Thereafter, replace the medium once every 2 days.

[0059] 2.7. When the cells grow to about 80% fusion, digest the cells with trypsin digestion solution, place in a 37℃ constant temperature box for 3-5 minutes, and when the cells shrink under an inverted microscope, shake the culture bottle horizontally to make most of the cells fall off, and add an appropriate amount of culture medium containing 10% fetal bovine serum to terminate digestion. Then transfer to a centrifuge tube for centrifugation, resuspend the cells with α-MEM culture medium containing 10% fetal bovine serum, and subculture at a ratio of 1:3.

[0060] 2.8. When the second generation cells grew to 60%-70% confluence, recombinant lentivirus carrying the green fluorescent protein gene was added and incubated for 48 hours, then digested with trypsin digestion solution for 5 minutes, and terminated with α-MEM culture medium containing 10% fetal bovine serum. The cell suspension was collected and the supernatant was removed by centrifugation.

[0061] 2.9. Resuspend the cells in fresh culture medium and continue culturing. Observe the fluorescence expression after 48 hours.

[0062] 2.10. Digest, centrifuge, resuspend and adjust the cell concentration to 10 cells / mL. Add 100 μL of cell suspension to each well of a 96-well culture plate. Observe and select the wells expressing green fluorescent protein and single cells under an inverted fluorescence microscope, mark them and expand the culture for later use.

[0063] 3. Cultivation of neurons

[0064] 3.1. Neonatal rats (within 24 hours of birth) were killed by cervical dislocation and disinfected with 75% ethanol. The skull and brain were then separated in a sterile culture dish. The separated whole brain was then placed in a culture dish containing sterile 4°C PBS solution. The brain stem and hippocampus were carefully removed under a stereomicroscope, the meninges and small blood vessels on the brain tissue were separated, and the separated cerebral cortex was transferred to another sterile culture dish.

[0065] 3.2. Use sterile ophthalmic scissors to quickly cut the isolated cerebral cortex into small pieces, add trypsin digestion solution, and digest in a 37°C constant temperature incubator for 15 minutes, blowing twice with a pipette during the process. Observe under the microscope, and after a large number of cells are digested, add α-MEM medium containing 10% fetal bovine serum to stop digestion.

[0066] 3.3. Use a pipette to gently blow a few times, absorb the upper single cell suspension, drop it into a 100μm mesh filter, collect the suspension and put it into a 15ml sterile centrifuge tube, and then centrifuge it at low temperature (4℃, 800 rpm, 5 minutes). Discard the supernatant, add α-MEM culture medium to resuspend the cells at the bottom. Let it stand for 5 minutes, and transfer the cell suspension to another centrifuge tube for use.

[0067] 4. Co-culture of green fluorescent protein-labeled bone marrow mesenchymal stem cells and neurons

[0068] 4.1. The experiment was divided into contact co-culture group and double-layer co-culture group.

[0069] 4.2. Contact co-culture: Mix the green fluorescent protein-labeled bone marrow mesenchymal stem cells obtained in 2.9 with the neurons obtained in 3.3 at a cell ratio of 1:3 to a cell concentration of 5×10 6 / ml, extract 0.2ml and slowly inoculate it at multiple points on the polylactic acid-glycolic acid porous cell scaffold treated in 1.4; 1 hour later, supplement with α-MEM culture medium containing 10% fetal bovine serum until the entire polylactic acid-glycolic acid porous cell scaffold is covered; 3 hours later, change the medium, discard the culture medium and non-adherent cells, add neuron-specific culture medium (containing 1% B27 and 0.3mg / ml cytarabine) and continue culturing.

[0070] 4.3. Double-layer co-culture: The green fluorescent protein-labeled bone marrow mesenchymal stem cells obtained in 2.9 and the neurons obtained in 3.3 were added to the upper and lower layers of the Transwell culture plate wells respectively (the cell inoculation amount was 1:3), and α-MEM culture medium containing 10% fetal bovine serum was added to mix the upper and lower layers of liquid; after 4 hours, the entire volume of the medium was replaced, and a neuron-specific culture medium (containing 1% B27 and 0.3 mg / ml cytarabine) was added to continue the culture.

[0071] 4.4. The co-cultured cells of the above two groups were replaced with half the volume of neuron-specific culture medium (containing 1% B27 and 0.3 mg / ml cytarabine) every 2 to 3 days, and the cells were collected on day 7.

[0072] 5. Immunofluorescence staining

[0073] 5.1. The polylactic-co-glycolic acid porous cell scaffolds of the contact co-culture group in 4.4 were frozen and sectioned (thickness 0.8 μm) and the cell slides of the upper layer of bone marrow mesenchymal stem cells of the double-layer co-culture group were incubated with 1% Triton X-100 at 37° C. for 30 minutes.

[0074] 5.2. Rinse with PBS for 5 minutes x 3 times.

[0075] 5.3. Incubate with 10% normal goat serum at 37°C for 1 hour without washing.

[0076] 5.4. Add primary antibody (mouse anti-rat NeuN antibody) (1:100) and incubate at 4°C overnight.

[0077] 5.5. Rinse with PBS for 5 minutes x 3 times.

[0078] 5.6. Add Cy3-labeled goat anti-mouse secondary antibody (1:200) and incubate at 37°C for 1 hour.

[0079] 5.7. Rinse with PBS for 5 minutes x 3 times.

[0080] 5.8. Add anti-fluorescence quencher and cover with a coverslip.

[0081] 5.9. Observe and take photos under a fluorescence microscope.

[0082] 6. Quantitative PCR to determine Notch-1 mRNA expression

[0083] 6.1. Take out the polylactic acid-glycolic acid porous cell scaffolds from the contact co-culture group in 4.4, rinse them twice with PBS, and then digest them in trypsin digestion solution at 37°C for 6 minutes. Gently shake to promote cell detachment, terminate the digestion with α-MEM culture medium containing 10% fetal bovine serum, and collect the mixed cell suspension.

[0084] 6.2. Centrifuge the mixed cells at 1000 rpm for 5 minutes, remove the supernatant, resuspend the cells in PBS, and obtain bone marrow mesenchymal stem cells labeled with green fluorescence by flow cytometry.

[0085] 6.3. Centrifuge at 1000 rpm for 5 minutes, remove the supernatant, and obtain the precipitate of bone marrow mesenchymal stem cells labeled with green fluorescence.

[0086] 6.4. Total RNA was extracted from the upper layer of the double-layer co-culture group in 4.4 and the bone marrow mesenchymal stem cells isolated from the contact co-culture group in 6.3 using the TRIZOL kit, and the RNA concentration was determined using a spectrophotometer.

[0087] 6.5. Reverse transcription and cDNA synthesis

[0088] 6.5.1. Prepare the reaction system (20 μl) on ice according to the instructions of the reverse transcription kit: 5×RT Buffer (4 μl), dNTP (2 μl), Super-RI (0.5 μl), RT-Enhance (0.5 μl), Oligo(dT)18 (1 μl), ReverTra Ace (1 μl), RNA+RNase Free H2O (11 μl, including 2 μg RNA).

[0089] 6.5.2. Place the prepared reaction system into the PCR analyzer and set the reaction conditions: 30℃ (10min) → 42℃ (20min) → 99℃ (5min) → 4℃ (5min).

[0090] 6.5.3. Label the samples that have been reverse transcribed into cDNA and place them in a -20℃ refrigerator for future use.

[0091] 6.6 Quantitative PCR

[0092] 6.6.1. Design primers:

[0093] Notch-1-F (SEQ ID NO. 1): 5'-CTGGTCAGGGAAATCGTG-3'.

[0094] Notch-1-R (SEQ ID NO. 2): 5'-TGGGCAGTGGCAGATGTAG-3'.

[0095] β-action-F (SEQ ID NO. 3): 5'-CGTTGACATCCGTAAAGAC-3'.

[0096] β-action-R (SEQ ID NO. 4): 5'-TGGAAGGTGGACAGTGAG-3'.

[0097] 6.6.2. Prepare the reaction system (20 μl) according to the instructions of QuantiNoveTMSYBR@Green PCR Kit: RNase Free H2O (5 μl), QN ROX Reference Dye (2 μl), SYBR Green PCR Master Mix (10 μl), Primer F (0.5 μl), Primer R (0.5 μl), cDNA (2 μl), and add to 8 tube strips.

[0098] 6.6.3. Place the 8-tube strip into the fluorescence quantitative PCR instrument and set the reaction conditions: pre-denaturation at 95°C (2 min), denaturation at 95°C (30 s) → annealing at 58°C (30 s) → extension at 72°C (1 min) for 35 cycles, continue extension at 72°C (1 min), and terminate (4°C).

[0099] 6.6.4. Import the Ct values ​​automatically generated by the computer into the Excel table and use 2 -ΔΔCt The data were analyzed by relative quantitative method.

[0100] 7. Protein immunoblotting

[0101] 7.1. The bone marrow mesenchymal stem cells separated from the contact co-culture group in 6.3 and the bone marrow mesenchymal stem cells from the upper layer of the double-layer co-culture group in 4.4 were lysed with protein lysis buffer, centrifuged at 12,000 rpm for 5 minutes at 4°C, and the supernatant was collected to determine the protein concentration using the BCA method.

[0102] 7.2. Add the protein supernatant obtained in 7.1 into the loading buffer in proportion and boil to denature the protein.

[0103] 7.3. Separate the proteins by SDS polyacrylamide gel electrophoresis and transfer them to PVDF membrane.

[0104] 7.4. Rinse the PVDF membrane in PBST solution for 10 minutes to remove the residual SDS on the membrane as much as possible, and then place it in a shaker containing 5% skimmed milk powder prepared with PBST for 2 hours.

[0105] 7.5. Place the PVDF membrane in 5% skim milk powder prepared in PBST, add primary antibodies of appropriate concentration (β-actin at 1:1000; Notch-1 at 1:500), and incubate at 4°C overnight.

[0106] 7.6. Wash the PVDF membrane with appropriate amount of PBST for 4 times, 10 minutes each time.

[0107] 7.7. Place the PVDF membrane in 5% skimmed milk powder prepared in PBST, add appropriate concentration of secondary antibody (HRP-labeled goat anti-mouse IgG, 1:2000), and incubate on a shaker at room temperature for 1 hour.

[0108] 7.8. Wash the PVDF membrane with appropriate amount of PBST for 4 times, 10 minutes each time.

[0109] 7.9. After scanning the experimental strips with a scanner, use Image J to calculate the grayscale value of the image.

[0110] 8. Results

[0111] 8.1. The bone marrow mesenchymal stem cells in subculture have good growth status and are relatively uniform in shape, mainly spindle-shaped, but also round, triangular and polygonal ( Figure 1 ).

[0112] 8.2. After mesenchymal stem cells were labeled with recombinant lentivirus carrying the green fluorescent protein gene, strong green fluorescence could be observed under a fluorescence microscope, indicating a good labeling effect ( Figure 2 ).

[0113] 8.3. On the 7th day of primary cerebral cortical neuron culture, the cell bodies were mainly round, oval, and spindle-shaped, and the processes were slender and began to interweave to form a network, indicating that the cells were growing well ( Figure 3 ).

[0114] 8.4. The polylactic-co-glycolic acid porous cell scaffold is honeycomb-shaped, with crisscrossing internal pores and good connectivity ( Figure 4 ).

[0115] 8.5. GFP-labeled bone marrow mesenchymal stem cells and neurons were seeded on polylactic-co-glycolic acid porous cell scaffolds. Both cells grew well ( Figure 5 ).

[0116] 8.6. Compared with the BMSCs in the double-layer co-culture group, the proportion of BMSCs expressing NeuN (neuronal marker protein) in the contact co-culture group increased significantly ( Figure 6 ).

[0117] 8.7. The Notch signaling pathway is an important pathway that mediates direct contact signals between adjacent cells, and its function in regulating cell proliferation and differentiation involves almost all tissues and organs. The experimental results showed that compared with the bone marrow mesenchymal stem cells in the double-layer co-culture group, the Notch-1 mRNA and protein expression of the bone marrow mesenchymal stem cells in the contact co-culture group was significantly decreased ( Figure 7 ).

[0118] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0119] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for constructing a three-dimensional contact co-culture system of bone marrow mesenchymal stem cells and neurons, characterized in that: The following steps are involved: S1, dissolving lactic acid and glycolic acid in an organic solvent, mixing with a porogen, and then placing in a mold for forming, demoulding, removing the sodium chloride porogen, and drying to obtain a polylactic acid-glycolic acid porous cell scaffold, cutting the polylactic acid-glycolic acid porous cell scaffold into a specific size, and then disinfecting, cleaning, and soaking in polylysine in sequence, placing the treated polylactic acid-glycolic acid porous cell scaffold in a first culture plate, drying under sterile conditions, and then adding α-MEM into the wells to soak overnight, and transferring the treated polylactic acid-glycolic acid porous cell scaffold to a second culture plate to dry; S2, preparing a cell suspension of bone marrow mesenchymal stem cells labeled with green fluorescent protein; S3. Preparation of cell suspension of primary neurons; S4. Mix the cell suspension of bone marrow mesenchymal stem cells labeled with green fluorescent protein with the cell suspension of neurons, and then inject them into the polylactic acid-glycolic acid porous cell scaffold in the second culture plate for culture at multiple points. After 3-4 hours, change the medium, discard the culture medium and non-adherent cells, add a neuron-specific culture medium containing cytarabine and continue to culture for 6-7 days to obtain a three-dimensional contact co-culture system of bone marrow mesenchymal stem cells and neurons.

2. The method for constructing a three-dimensional contact co-culture system of bone marrow mesenchymal stem cells and neurons according to claim 1, characterized in that: In S1, the molar ratio of lactic acid to glycolic acid is 80:

20.

3. The method for constructing a three-dimensional contact co-culture system of bone marrow mesenchymal stem cells and neurons according to claim 1, characterized in that: In S1, the organic solvent is dichloromethane, and the mass volume ratio of lactic acid and glycolic acid to dichloromethane is 1g:6-10ml.

4. The method for constructing a three-dimensional contact co-culture system of bone marrow mesenchymal stem cells and neurons according to claim 1, characterized in that: In S1, the porogen is sodium chloride particles with a particle size of 180 to 250 μm.

5. The method for constructing a three-dimensional contact co-culture system of bone marrow mesenchymal stem cells and neurons according to claim 1, characterized in that: In S1, the mass ratio of lactic acid and glycolic acid to the porogen is 1:15 to 1:

22.

6. The method for constructing a three-dimensional contact co-culture system of bone marrow mesenchymal stem cells and neurons according to claim 1, characterized in that: In S2, preparing a cell suspension of bone marrow mesenchymal stem cells labeled with green fluorescent protein comprises the following steps: A1. Isolate the bilateral tibiae and femurs of 1-month-old rats, and flush the bone marrow cavity with phosphate buffer to obtain a cell suspension; A2, adding the cell suspension described in A1 onto the rat lymphocyte separation solution, and collecting the interface cell layer by centrifugation; A3, resuspending the cells of the interface cell layer described in A2 in α-MEM medium containing 10% fetal bovine serum, inoculating them in a culture flask and culturing them for 2 hours; A4: Replace the entire volume of medium to remove non-adherent cells and impurities in A3, and replace the medium every 2 to 3 days thereafter; When the cells obtained from A5 and A4 reached about 80% confluence, the cells were digested with trypsin, then centrifuged and resuspended in α-MEM medium containing 10% fetal bovine serum for further subculture; When the passaged A6 and A5 cells reached 60%-70% confluence, recombinant lentivirus carrying the green fluorescent protein gene was added and incubated for 48 hours; The cells cultured in A7 and A6 were digested with trypsin digestion solution, then centrifuged to remove the supernatant, resuspended in fresh culture medium, and continued to be cultured, and the fluorescence expression was observed in 24-48 hours; After digestion, the cultured cells of A8 and A7 were resuspended and the cell concentration was adjusted to 10 cells / mL. 100 μL of cell suspension was added to each well of the 96-well plate. The wells expressing GFP and single cells were observed and selected under an inverted fluorescence microscope for labeling and expansion of culture.

7. The method for constructing a three-dimensional contact co-culture system of bone marrow mesenchymal stem cells and neurons according to claim 1, characterized in that: In S3, preparing a cell suspension of primary neurons includes the following steps: B1. Isolate the cerebral cortex of newborn rats within 24 hours of birth and cut it into pieces. Add digestive enzyme solution and place it in a 37°C incubator for digestion. During the digestion, pipette and perfuse 1-2 times. B2, adding DMEM culture medium containing 10% fetal bovine serum to the brain tissue of B1 to terminate digestion and prepare a suspension, and dropping it into a cell sieve with a pore size of 100 μm to obtain a first cell suspension; B3. After centrifuging the first cell suspension of B2, the supernatant was removed, and the precipitate was resuspended in α-MEM culture medium containing 10% fetal bovine serum, and allowed to stand for 5-10 minutes to remove fibroblasts to obtain a second cell suspension, i.e., the cell suspension of primary neurons.

8. The method for constructing a three-dimensional contact co-culture system of bone marrow mesenchymal stem cells and neurons according to claim 1, characterized in that: In S4, the ratio of green fluorescent protein-labeled bone marrow mesenchymal stem cells to neurons is 1:2-4.

9. The method for constructing a three-dimensional contact co-culture system of bone marrow mesenchymal stem cells and neurons according to claim 1, characterized in that: In S4, the seeding density of green fluorescent protein-labeled bone marrow mesenchymal stem cells and neurons on the polylactic-co-glycolic acid porous cell scaffold was 2×10 6 / cm 3 ~5×10 6 / cm 3 .

10. The three-dimensional contact co-culture system of bone marrow mesenchymal stem cells and neurons constructed by the construction method according to any one of claims 1 to 9.

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