CN106676069B - Experimental method for promoting NG2 cells to differentiate into nerve cells by TSP4 - Google Patents
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Abstract
The invention discloses an experimental method for promoting NG2 cells to be differentiated into nerve cells by TSP4, which comprises the following steps: culturing cells; construction of plasmid pXJ40-myc-TSP 4; detecting Western blotting; immunofluorescence; extracting total RNA and carrying out real-time fluorescence quantitative PCR; and (5) statistical treatment. The invention aims to explore the role of TSP4 in the differentiation of NG2 cells into neuronal cells and elucidate the molecular mechanism of TSP4 in regulating the differentiation of NG2 neurons. TSP4 can promote the transdifferentiation of NG2 cells into nerve cells by inhibiting the activity of MAPK/ERK signaling pathway. The invention provides an experimental means, which can effectively induce NG2 cells to differentiate into nerve cells after TSP4 is over-expressed in NG2 cells. The invention can provide important basis for the TSP4 gene applied to the treatment of spinal cord injury and neurodegenerative diseases.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to an experimental method for promoting differentiation of NG2 cells into nerve cells by TSP 4.
Background
With the development of economic levels of countries in the world, the incidence of spinal cord injury tends to increase year by year. Spinal cord injury is the most serious complication of spinal cord injury, often leading to severe dysfunction of the limb below the injured segment. Nerve cells belong to terminally differentiated cells, have weak proliferation ability, and are difficult to regenerate when damaged. The spinal cord injury not only can bring serious physical and psychological trauma to patients, but also can cause huge economic burden to the whole society, so that the prevention, treatment and rehabilitation aiming at the spinal cord injury become a big problem in the medical field at present.
In the mammalian central nervous system, there is a kind of colloid precursor cell, also called NG2 cell because of its property of expressing chondroitin sulfate proteoglycan on its surface. NG2 cells are widely distributed in the developing and mature central nervous system, and can show the characteristics of morphological diversity, differentiation plasticity and functional diversity under specific physiological and pathological conditions. Previous work proves that NG2 cells can be transformed into neuron cells under certain induction conditions, and the condition of the disease is obviously improved after the induced NG2 cells are transplanted into a spinal cord injury mouse, so that the eosin is brought to clinical treatment of nerve injury diseases such as spinal cord injury.
Thrombospondin 4 (TSP 4) belongs to thrombospondin family, has multiple biological functions, and the thrombospondin family members are one kind of cell-cell and cell-matrix adhesion mediating glucoprotein. Girard et al demonstrated that TSP4 is deficient in fetal as well as adult murine brain models, and that its neuronal cell migration capacity is significantly impaired. They are closely related to cell proliferation, migration, adhesion and linkage, and also produce inflammatory responses to central nervous system injury. Kim et al demonstrated that TSP4 can cause presynaptic hypersensitivity and neuropathic pain in the spinal cord following peripheral nerve injury. These results suggest that TSP4 is closely related to the occurrence and development of nerve cells. At present, it is not clear how TSP4 regulates oligodendrocyte precursor cells (NG2 cells) to differentiate into neuronal cells, and promotes the regeneration of neurons. It has been reported that TSP4 plays an important role in diseases such as spinal sensitization, tendon formation and neuropathic pain, but its role in neural differentiation is rarely reported at present.
In addition to another gene NMEI discovered in 2012, there is no other gene currently used for inducing the differentiation of NG2 cells into nerve cells. The invention aims to establish a method for inducing oligodendrocyte precursor cells-NG 2 cells to differentiate into nerve cells, and provides help for TSP4 protein used for treating spinal cord injury.
Disclosure of Invention
In order to overcome the defects in the prior art, the NG2 cell is difficult to differentiate into nerve cells in vitro, and the nerve cells are further applied to the treatment of spinal cord injury. The invention provides an experimental method, which can effectively induce NG2 cells to differentiate into nerve cells after TSP4 is over-expressed in NG2 cells.
The technical scheme is as follows:
an experimental method for promoting differentiation of NG2 cells into nerve cells by TSP4 comprises the following steps:
NG2 cells were cultured in DMEM/F12 medium containing 2% fetal bovine serum, 100U/mL penicillin, 100. mu.g/mL streptomycin, B27supplement, N2supplement in 5% CO2The constant temperature incubator is used for incubation at 37 ℃;
According to the mRNA sequence NM-017133.1 of TSP4 gene in GenBank, DNAMAN8.0 software is used for designing primers, the TSP4 gene is amplified from a rat cDNA library, the amplified fragment is connected to a pXJ40-myc eukaryotic expression vector, TOP10 competent cells are transformed, an ampicillin resistant LB agar plate is used for screening monoclonal colonies, bacteria are shaken and plasmids are extracted, plasmid restriction enzyme digestion verification and electrophoresis analysis are carried out, and the positive plasmids are sent to Suzhou Jinzhi Biotech company for sequencing;
Adding a cell lysate to lyse cells, extracting proteins, performing protein quantification by using a Bradford method, adding a 5 xSDS-PAGE sample loading buffer solution, boiling for 5 minutes at 100 ℃, loading, performing 80V electrophoresis for 30 minutes, performing 120V electrophoresis for 50 minutes, performing 390mA membrane rotation for 70 minutes, sealing 5% skimmed milk for 1 hour, performing primary antibody at 4 ℃ overnight, rinsing for 3 times by TBST, performing each time for 5 minutes, adding a corresponding secondary antibody for incubation for 1 hour, rinsing for 3 times by TBST, performing each time for 5 minutes, performing chemiluminescence by an Amersham Imager600 instrument, and performing gray scale analysis by using ImageJ1.48 software;
Adding the transfected NG2 cells into a 12-hole plate paved with a cell slide, and performing corresponding treatment after cell slide: cells were fixed in 4% paraformaldehyde for 30 min and rinsed 3 times in PBS for 5 min each. 0.1% Triton X-100 was permeabilized for 10 minutes at room temperature and rinsed 3 times with PBS for 5 minutes each. Blocking with 3% BSA at room temperature for 1 hour, rinsing 3 times with PBS for 5 minutes each. Adding primary anti-MAP 2 and NF 2004 ℃ to incubate overnight, rinsing with PBS for 3 times and 5 minutes each time, then adding FITC-labeled secondary antibody, incubating for 2 hours, counterstaining with DAPI for 5 minutes, rinsing with PBS for 3 times and 5 minutes each time, sealing with 90% glycerol, and observing and taking a picture with a laser confocal microscope;
Trizol reagent extracts total RNA of cells, carries out reverse transcription kit synthesis of cDNA, carries out real-time fluorescence quantitative PCR by taking the cDNA as a template according to the instruction, detects the expression of a target gene, and has the following primer sequences of GAPDH 5'-GGTCGG TGT GAA CGG ATT TG-3', 5'-GCT TCC CAT TCT CAG CCT TGA-3', NF200, 5'-ACCTATACC CGA ATG CCT TCT-3', 5'-AGA AGC ACT TGG TTT TAT TGC AC-3', NeuN 5'-CAGGCC TCA GAA ACA CAC AA-3', 5'-AGC ACC AGTAGAAAT GGA TGA-3', Tuj1, 5'-TGA CGA GCATGG CAT AGA CC-3', 5'-TGT TGC CAG CAC CAC TCT GA-3', neuroD1, 5'-CTT GGC CAA GAACTA TAT CTG G-3' and 5'-GGA GTA GGG ATG CAC CGG GAA-3'. the reaction system comprises EvaGreen 2 × qPCR MasterMix 10 muL, upstream primer 0.6 muL, downstream primer 0.6 muL, template cDNA2 muL and double distilled water 6.8 muL, and the reaction program comprises the steps of 95 ℃ 10 minutes, 95 ℃ 15 seconds, 60 ℃ 1 minute, and total 40 cycles, and the relative expression of the gene is 2 muL-ΔΔCtCalculating, using rat GAPDH as reference gene;
Statistical processing was performed using SPSS 18.0 statistical software. The mean value is represented as +/-standard deviation, the comparison of observation indexes among groups adopts one-factor variance analysis and a minimum significant difference method, and the two sides P of the detection level alpha value is less than 0.05.
Further, the immunoblotting result of step 3 shows that TSP4 can promote the expression of NG2 cell neuron differentiation marker proteins NF200, Tuj1 and MAP2 in the experimental group in which the TSP4 gene is overexpressed, compared with the control group; compared with a control group, TSP4 of an experimental group over-expressing TSP4 gene can promote activation of an ERK signal pathway of NG2 cells, and has no significant influence on p38 and AKT signal pathways; when the ERK signaling pathway was blocked with U0126, the expression of the neuronal marker protein NeuN of NG2 cells was increased.
Further, the immunofluorescence staining result in step 4 shows that the TSP4 can promote the expression of the NG2 cell neuron differentiation marker proteins NF200 and MAP2 in the experimental group in which the TSP4 gene is overexpressed, compared with the control group.
Further, the fluorescent quantitative PCR result of step 5 shows that TSP4 can promote the expression of NG2 cell neuron differentiation marker genes NeuN, NF200, Tuj1 and NeuroD1 in the experimental group over-expressing the TSP4 gene compared with the control group; when the ERK signaling pathway was blocked with U0126, the expression of the neuronal marker genes NeuN, NF200, Tuj1 and NeuroD1 of NG2 cells was significantly increased.
The experimental result shows that TSP4 can promote the transdifferentiation of NG2 cells to nerve cells by inhibiting the activity of MAPK/ERK signaling pathway. Western blotting and real-time PCR results show that in an experiment group of over-expressing TSP4, the expressions of neuron differentiation marker proteins NeuN, NF200 and Tuj-1 are all up-regulated, and meanwhile, the phosphorylation level of ERK is reduced. After the ERK signal channel specific blocker is added, Western blotting shows that the expression level of the neural differentiation marker protein NeuN is up-regulated, and real-time PCR results simultaneously prove that the expression levels of a plurality of neural differentiation marker genes are increased after the ERK blocker is added. The above results suggest that TSP4 can promote the differentiation of NG2 cells into nerve cells by inhibiting the activity of MAPK/ERK signaling pathway. The invention provides theoretical reference for clinical treatment of spinal cord injury. The virus expression vector using TSP4, TSP4 eukaryotic cell expression plasmid or direct TSP4 protein to induce NG2 cell to differentiate into nerve cell, and the application in treating nerve degenerative diseases or spinal cord injury are protected by the present patent.
Drawings
FIG. 1 shows that TSP4 has a novel function of promoting neural differentiation of NG2 cells; wherein, the Western blot detection of endogenous and myc tag fused TSP4 expression 2 days after TSP4(pXJ40-myc/TSP4) is shown in figure 1A, and the immunofluorescence detection of the expression of NG2 cell neural markers NF200 and MAP2 (scale bar: 100 μm) is shown in figure 1B after TSP4 is over-expressed.
Figure 2 is a graph showing that TSP4 is capable of increasing expression of neural differentiation markers; wherein, fig. 2A shows Western blot detection of expression of neuronal differentiation markers NF200, Tuj1 and NeuN, β -actin is used as an internal reference, fig. 2B-fig. 2D show quantitative analysis results of NeuN, NF200, Tuj1 and NeuroD1 proteins, respectively, fig. 2E shows real-time fluorescence quantitative PCR detection of expression of gene level of the neuronal markers, and GAPDH is used as an internal reference gene. Values are taken as the mean of triplicates ± standard deviation. P < 0.05, P < 0.01 and P < 0.001, compared to control;
FIG. 3 is a graph showing that TSP4 mediates neural differentiation of NG2 cells by inhibiting the MAPK/ERK signaling pathway, but not the MAPK/p38 and PI3K/AKT signaling pathways; among them, FIG. 3A shows that TSP4 can inhibit activation of ERK signaling pathway without affecting PI3K/AKT and MAPK/p38 signaling pathways, and FIGS. 3B-3D show quantitative analysis of changes in p-ERK, p-AKT and p-p38 expression levels, respectively. Values are taken as the mean of triplicates ± standard deviation. P < 0.001, compared to control.
FIG. 4 is a graph showing that inhibition of MAPK/ERK signaling pathway promotes neural differentiation of NG2 cells, wherein FIG. 4A shows that after treatment with different concentrations of ERK signaling pathway blocker U0126 for 1 day, p-ERK levels are significantly reduced as detected by 20 and 30 μ M U0126, FIG. 4B shows that after ERK signaling pathway is blocked, the expression of the neural marker NeuN is significantly increased compared with a control, FIG. 4C shows that the U0126 treatment group is increased by 2.5 times compared with the control group as a result of quantitative analysis, and FIG. 4D shows that real-time fluorescence quantitative PCR detects the expression of different neural markers. Values are taken as the mean of triplicates ± standard deviation. P < 0.05, P < 0.01, compared to control.
Detailed Description
The technical solutions of the present invention will be described in further detail with reference to the accompanying drawings and the detailed description.
1 materials and methods
1.1 materials
Rat NG2 cells were isolated and cultured in this laboratory; DMEM/F12 medium was purchased from HyClone, Fetal Bovine Serum (FBS) was purchased from Gibco, RIPA cell lysis, protease inhibitors, phosphatase inhibitors, penicillin, streptomycin, trypsin were purchased from Shanghai Bin Yuntan Biotech Ltd; TSP4 antibody was purchased from R & D system; NeuN, NF200 antibodies were purchased from emdmilliplore. MAP2, AKT, p-AKT, ERK2, p-ERK1/2, p-38, and β -actin antibodies were purchased from CST Inc. Specific inhibitor U0126 was purchased from Sigma. The Trizol reagent is a TaKaRa product. The reverse transcription kit and the real-time fluorescent quantitative PCR kit are purchased from ABM company. Lipofectamine 2000 was purchased from Invitrogen.
1.2 cell culture
NG2 cells were cultured in DMEM/F12(Dulbecco Modified Eagle Medium, HyClone) Medium containing 2% fetal bovine serum, 100U/mL penicillin, 100ug/mL streptomycin, B27supplement (50 ×, Gibco), N2supplement (100 ×, Gibco) in 5% CO237 ℃ incubator.
1.3 construction of expression plasmid pXJ40-myc-TSP4
According to the mRNA sequence (NM-017133.1) of TSP4 gene in GenBank, DNAMAN8.0 software is used for designing primers, the TSP4 gene is amplified from a rat cDNA library, the amplified fragment is connected to a pXJ40-myc eukaryotic expression vector, competent TOP10 is transformed, an ampicillin resistant LB agar plate is used for screening monoclonal colonies, bacteria are shaken and plasmids are extracted, and plasmid restriction enzyme digestion verification and electrophoretic analysis are carried out. The positive plasmids were sequenced by the Biotech company Jinzhi, Suzhou.
1.4 Western blot detection
Adding RIPA lysate to lyse the cells, extracting protein, and performing protein quantification by using Bradford method. Adding 5 xSDS-PAGE sample buffer, boiling at 100 deg.C for 5 min, loading, running at 80V for 30 min, running at 120V for 50 min, rotating membrane at 390mA for 70 min, blocking with 5% skimmed milk for 1 hr, and standing at 4 deg.C overnight. Rinse 3 times with TBST for 5 minutes each, add corresponding secondary antibody to incubate for 1 hour, and rinse 3 times with TBST for 5 minutes each. The Amersham Imager600 instrument was chemiluminescent and analyzed for gray scale using ImageJ1.48 software (National Institutes of Health, USA).
1.5 immunofluorescence
Adding the transfected NG2+ cells into a 12-hole plate paved with a cell slide, and performing corresponding treatment after cell slide: cells were fixed in 4% paraformaldehyde for 30 min and rinsed 3 times in PBS for 5 min each. 0.1% Triton X-100 was permeabilized for 10 minutes at room temperature and rinsed 3 times with PBS for 5 minutes each. Blocking with 3% BSA at room temperature for 1h, rinsing 3 times with PBS for 5 minutes each. Primary anti-MAP 2 (1: 100) and NF200 (1: 100) were added and incubated overnight at 4 ℃ and rinsed 3 times with PBS for 5 minutes each. FITC-labeled secondary antibody was then added, incubated for 2h, DAPI counterstained for 5 minutes, rinsed 3 times with PBS for 5 minutes each, 90% glycerol mounted, and photographed by confocal laser observation.
1.6 Total RNA extraction and real-time fluorescent quantitative PCR
Trizol reagent extracts total RNA of cells, carries out reverse transcription kit synthesis cDNA, and carries out real-time fluorescence quantitative PCR by taking the cDNA as a template, and detects the expression of a target gene, wherein the primer sequence is GAPDH 5'-GGTCGG TGT GAA CGG ATT TG-3', 5'-GCT TCC CAT TCT CAG CCT TGA-3', NF200, 5'-ACC TATACC CGA ATG CCT TCT-3', 5'-AGA AGC ACT TGG TTT TAT TGC AC-3', NeuN 5'-CAG GCCTCA GAA ACA CAC AA-3', 5'-AGC ACC AGTAGA AAT GGA TGA-3', Tuj1, 5'-TGA CGA GCATGG CAT AGA CC-3', 5'-TGT TGC CAG CAC CAC TCT GA-3', neuroD1, 5'-CTT GGC CAA GAACTA TAT CTG G-3' and 5'-GGA GTA GGG ATG CAC CGG GAA-3' the reaction system is EvaGreen 2 × qPCR MasterMix 10 muL, an upstream primer 0.6 muL, a downstream primer 0.6 muL, template cDNA2 muL and double distilled water 6.8 muL, the reaction program is 95 ℃ for 10 minutes, 95 ℃ for 15 seconds, 60 ℃ for 1 minute, and the relative expression amount of the gene is 2-ΔΔCtMethod calculation, rat GAPDH was used as the reference gene.
1.7 statistical treatment
Statistical processing was performed using SPSS 18.0 statistical software. The mean value plus or minus standard deviation is used for representing, single-factor variance analysis and the minimum significant difference method are adopted for comparing observation indexes of all groups, and the two sides P of the detection level alpha value is less than 0.05.
2 results
2.1 TSP4 has the function of promoting the neural differentiation of NG2 cells
To understand the effect of TSP4 on NG2 cell neural differentiation, a myc-tagged TSP4 expression plasmid was transfected into NG2 cells, and its expression was detected with TSP4 and myc-tag antibodies, which demonstrated that TSP4 was overexpressed in NG2 cells (fig. 1A). We next found that cells over-expressing TSP4 exhibited a neuronal-like morphology (fig. 1B). This result suggests that TSP4 may play a certain promoting role in neural differentiation of NG2 cells.
2.2 TSP4 Induction of NG2 cell neural marker protein expression
To explore the effect of TSP4 on NG2 cell differentiation into neurons, we seeded NG2 cells in 6-well plates and total protein was extracted after 2 days when the cells were fused to 90% -95% Lipofectamine 2000 transfected cells. Western blotting was used to detect the expression of the neuronal markers NeuN, NF200 and Tuj-1. The results showed that the protein expression levels of Tuj-1, NF200 and NeuN of the experimental group expressing TSP4 were significantly up-regulated compared to the control group (fig. 2A). The expression level of NeuN in the experimental group over-expressing TSP4 was up-regulated by 79% (P < 0.01) (FIG. 2B), the expression level of NF200 was up-regulated by 261% (P < 0.01) (FIG. 2C), and the expression level of Tuj-1 was up-regulated by 165% (P < 0.001) (FIG. 2D).
Further, we verified the effect of TSP4 on neural differentiation markers from mRNA levels using real-time fluorescent quantitative PCR technology. Consistent with the Western blot results, mRNA expression of NF200, NeuN, Tuj1, and neural differentiation transcription factor NeuroD1 were all significantly upregulated (fig. 2E). The above results confirmed that TSP4 has a certain promoting effect on neural differentiation of NG2 cells.
2.3 TSP4 mediates neural differentiation of NG2 cells by inhibiting the MAPK/ERK signaling pathway, but not the MAPK/p38 and PI3K/AKT signaling pathways
MAPK/ERK, PI3K/AKT, and MAPK/p38 signaling pathways are known to play important roles in neural differentiation and axon growth. To explore the underlying mechanism by which TSP4 induces the neural differentiation phenotype, we examined whether these signaling pathways were affected by TSP4 (fig. 3). Immunoblot results revealed that the activity of MAPK/p38 and PI3K/AKT signaling homology was not significantly affected when TSP4 was overexpressed (FIGS. 3C and D). Compared with the control group, the activity of MAPK/ERK signaling pathway of the group over-expressing TSP4 is obviously inhibited (FIGS. 3A and B). This result suggests that TSP4 may mediate neural differentiation of NG2 by inhibiting the activity of the MAPK/ERK signaling pathway.
2.4 inhibition of the MAPK/ERK signaling pathway promotes neural differentiation of NG2 cells
From the above results, we found that TSP4 could inhibit the activity of MAPK/ERK signaling pathway, suggesting that MAPK/ERK signaling pathway may play a negative regulatory role in neural differentiation of NG2 cells. We therefore added U0126, a specific blocker of the MAPK/ERK signaling pathway, and observed whether U0126 had an effect on neural differentiation of NG2 cells. We treated cells with different concentrations of U0126(0, 10, 20, 30 μ M) for 24 hours first and extracted total protein, and the results showed that the level of ERK activation was significantly reduced after 20, 30 μ M of U0126 treatment (fig. 4A). When 20. mu. M U0126 was used to block the MAPK/ERK signaling pathway, the expression level of the neuronal marker NeuN was up-regulated by 257% (P < 0.01) (FIGS. 4B and C), which was statistically significant. Meanwhile, the real-time fluorescent quantitative PCR results also showed that the mRNA expression levels of the neural differentiation genes NF200, NeuN, Tuj1 and NeuroD1 of the U0126-treated group were significantly up-regulated compared to the control group (fig. 4D).
The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and it is within the scope of the present invention to use the viral expression vector of TSP4, the TSP4 eukaryotic cell expression plasmid or the TSP4 protein directly to induce NG2 cells to differentiate into nerve cells, and to use them for the treatment of human neurodegenerative diseases or spinal cord injury. Any simple changes or equivalent substitutions of technical solutions which can be obviously obtained by a person skilled in the art within the technical scope of the present disclosure fall within the protective scope of the present invention.
Claims (5)
1. An experimental method for promoting NG2 cells to differentiate into nerve cells by TSP4, which is characterized by comprising the following steps:
step 1, cell culture
NG2 cells were cultured in DMEM/F12 medium containing 2% fetal bovine serum, 100U/mL penicillin, 100. mu.g/mL streptomycin, B27supplement, N2supplement in a 37 ℃ incubator containing 5% CO 2;
step 2, construction and transfection of expression plasmid pXJ40-myc-TSP4
According to the mRNA sequence NM-017133.1 of TSP4 gene in GenBank, DNAMAN8.0 software is used for designing primers, the TSP4 gene is amplified from a rat cDNA library, the amplified fragment is connected to a pXJ40-myc eukaryotic expression vector, competent TOP10 is transformed, an ampicillin resistant LB agar plate is used for screening a monoclonal colony, bacteria are shaken and plasmids are extracted, plasmid restriction enzyme digestion verification and electrophoresis analysis are carried out, and the positive plasmids are sent to Suzhou Jinzhi Biotech company for sequencing; transfecting the constructed plasmid pXJ40-myc-TSP4 into the NG2 cells obtained in the step 1 to obtain NG2 cells over-expressing TSP 4;
step 3, Western blot detection
Adding RIPA lysate to lyse NG2 cells which are over-expressed with TSP4 and obtained in the step 2, extracting total protein, quantifying the protein by a Bradford method, adding 5 xSDS-PAGE loading buffer solution, boiling for 5 minutes at 100 ℃, loading the sample, carrying out 80V electrophoresis for 30 minutes, carrying out 120V electrophoresis for 50 minutes, transferring the membrane for 70 minutes at 390mA, sealing 5% skimmed milk for 1 hour, adding NF200, Tuj-1, MAP2, TSP4, p-ERK1/2, p-AKT, p-p38 and beta-actin primary antibody for overnight at 4 ℃, rinsing for 3 times by TBST, adding corresponding secondary antibody for incubation for 1 hour at 5 minutes each time, rinsing for 3 times by TBST for 5 minutes, carrying out chemiluminescence by an Amersham Imager600 instrument, and carrying out grey scale analysis by ImageJ1.48 software;
step 4, immunofluorescence
Adding the NG2 cells which are over-expressed with TSP4 and obtained in the step 2 into a 12-well plate paved with cell slides, and performing corresponding treatment after cell slide: cells were fixed with 4% paraformaldehyde for 30 min, rinsed 3 times with PBS for 5 min each; permeabilize 0.1% Triton X-100 for 10 minutes at room temperature, rinse 3 times with PBS for 5 minutes each time; blocking with 3% BSA at room temperature for 1 hour, rinsing with PBS 3 times for 5 minutes each; adding MAP2 and NF200 primary antibody, incubating overnight at 4 ℃, rinsing with PBS for 3 times and 5 minutes each time, adding FITC labeled secondary antibody, incubating for 2 hours, counterstaining with DAPI for 5 minutes, rinsing with PBS for 3 times and 5 minutes each time, sealing with 90% glycerol, and observing and photographing with a confocal laser microscope;
step 5, total RNA extraction and real-time fluorescence quantitative PCR
Extracting total RNA of NG2 cells over expressing TSP4 obtained in the step 2 by using Trizol reagent, synthesizing cDNA by using a reverse transcription kit, carrying out real-time fluorescence quantitative PCR by using the cDNA as a template by referring to the instruction, and detecting the expression of a target gene by using a primer sequence comprising GAPDH 5'-GGT CGG TGT GAA CGG ATT TG-3', 5'-GCT TCC CAT TCT CAG CCT TGA-3', NF200, 5'-ACC TAT ACC CGA ATG CCT TCT-3', 5'-AGA AGC ACT TGG TTT TAT TGC AC-3', NeuN 5'-CAG GCC TCA GAA ACA CAC AA-3', 5'-AGC ACCAGTAGAAATGGATGA-3', Tuj1, 5'-TGA CGA GCA TGG CAT AGA CC-3', 5'-TGT TGC CAG CAC CAC TCT GA-3', NeuroD1, 5'-CTT GGC CAA GAA CTA TAT CTG G-3' and 5'-GGA GTA GGG ATG CAC CGG GAA-3', wherein the reaction system comprises EvaGreen 2 × qPCR MasterMix 10 muL, an upstream primer 0.6 muL, a downstream primer 0.6 muL, a template cDNA2 muL and double distilled water 6.8 muL, the reaction program comprises the steps of 95 ℃ for 10 minutes, 95 ℃ for 15 seconds and 60 ℃ for 1 minute for 40 cycles, and the relative expression amount of the gene is 2-ΔΔCtCalculating, using rat GAPDH as reference gene;
step 6, statistical processing
Statistical processing is carried out by adopting SPSS 18.0 statistical software; the mean value plus or minus standard deviation is used for representing, single-factor variance analysis and the minimum significant difference method are adopted for comparing observation indexes of all groups, and the two sides P of the detection level alpha value is less than 0.05.
2. The experimental method for promoting the differentiation of NG2 cells into nerve cells by TSP4 as claimed in claim 1, wherein the immunofluorescence staining result of step 4 shows that TSP4 of the experimental group over-expressing TSP4 gene can promote the expression of NG2 cell neuron differentiation marker genes NF200 and MAP2 compared with the control group.
3. The experimental method for promoting the differentiation of NG2 cells into nerve cells by TSP4 as claimed in claim 1, wherein the immunoblotting result in step 3 shows that TSP4 can promote the expression of NG2 cell neuron differentiation marker proteins NF200, Tuj1 and MAP2 in the experimental group which overexpresses TSP4 gene compared with the control group.
4. The experimental method for promoting the differentiation of NG2 cells into nerve cells by TSP4 as claimed in claim 1, wherein the fluorescent quantitative PCR result in step 5 shows that TSP4 can promote the expression of NG2 cell neuron differentiation marker genes NeuN, NF200, Tuj1 and NeuroD1 in the experimental group which overexpresses TSP4 gene compared with the control group.
5. The experimental method for promoting the differentiation of NG2 cells into nerve cells by TSP4 as claimed in claim 1, wherein the immunoblotting result in step 3 shows that TSP4 can inhibit activation of ERK signaling pathway of NG2 cells compared with the control group, and has no significant effect on p38 and AKT signaling pathway in the experimental group in which TSP4 gene is overexpressed.
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