CN111662878A

CN111662878A - Application of NUDT12 gene in promoting growth and differentiation of human adipose-derived mesenchymal stem cells

Info

Publication number: CN111662878A
Application number: CN202010557705.9A
Authority: CN
Inventors: 王青
Original assignee: Shandong Rudai Biotechnology Co ltd
Current assignee: Shanghai Hanshi ark Biotechnology Co.,Ltd.
Priority date: 2020-06-18
Filing date: 2020-06-18
Publication date: 2020-09-15
Anticipated expiration: 2040-06-18
Also published as: CN111662878B

Abstract

The invention relates to an application of an NUDT12 gene in promoting growth and differentiation of human adipose-derived mesenchymal stem cells. After the NUDT12 gene is over-expressed in the adipose-derived stem cells, the induction efficiency of nerve cells and the proliferation speed of the induced nerve cells are remarkably improved, and the nerve cell identification has a good effect.

Description

Application of NUDT12 gene in promoting growth and differentiation of human adipose-derived mesenchymal stem cells

Technical Field

The invention relates to the field of stem cells, in particular to application of an NUDT12 gene in promoting growth and differentiation of human adipose-derived mesenchymal stem cells.

Background

With the development and maturation of molecular biology theory and technology, the application of tissue engineering to repair tissue defects has become a widely accepted development direction for scholars at home and abroad, wherein the selection of seed cells is the precondition and the key of tissue engineering. Adipose-derived mesenchymal stem cells are discovered in 2001, are rather rapid adult stem cells researched in recent years, are derived from mesoderm, and experiments prove that the adipose-derived mesenchymal stem cells have the potential of differentiating towards bones, cartilages, neurons, tendons, fat and the like, have high in-vivo content and high multiplication speed, have small damage to organisms due to primary material drawing, gradually replace bone marrow-derived mesenchymal stem cells with large trauma to the organisms, and are expected to become ideal seed cells in tissue engineering. The adipose-derived stem cells are easily obtained, can be obtained by a simple liposuction method, are sufficient in supply, can be repeatedly obtained, are less in damage and do not cause moral debate. The process of obtaining the stem cells by the liposuction method can also have a certain treatment effect on obesity and can also achieve the aims of beautifying and body building; in addition, since adipose-derived stem cells are rapidly proliferated, they are one of excellent seed cells for tissue engineering, and they are expected to be widely used in research and treatment in bone tissue engineering, adipose tissue engineering, repair of cartilage and muscle injuries, plastic surgery, etc. by being combined with a suitable three-dimensional biomaterial.

In recent years, with the development of stem cell biology and the study of molecular mechanisms of neurogenesis in adults, cell replacement therapy has drawn attention from researchers because it has the potential to cure neurodegenerative diseases. The adipose-derived mesenchymal stem cells have rich reserves in vivo and convenient acquisition, and become a research hotspot for treating neurodegenerative diseases by the stem cells.

The differentiation of ADSCs towards the nerve direction is carried out by the common method which comprises (1) the culture of nerve induction culture medium; (2) co-culturing; (3) gene transfection; (4) and (4) electrically stimulating. The induction culture of the nerve induction culture medium adopts a direct induction differentiation or step induction differentiation method, namely, the ADSCs are firstly trans-differentiated into Neural Stem Cells (NSCs) and then are differentiated towards the nerve cells. The culture medium is widely used for adding growth factors, and hormone proteins, small molecular compounds and the like are also added. Razavi et al first induced human ADSCs into neurotrophic factor-secreting cells, then made into cell microcapsules, co-cultured with neurospheres induced by human ADSCs at a ratio of 1:1, and finally the MAP2 and nestin expressed by the induced cells are significantly increased, while GFAP is decreased, indicating that co-culturing human ADSCs and neurotrophic factor-secreting cells promotes differentiation toward neurons, and the promotion is achieved by secreting neurotrophic factor-secreting cells BDNF, neurotrophic factors (NGF), ciliary neurotrophic factors (CNTF) and the like. In addition to adding various cytokines, hormones and small molecular compounds into a nerve culture medium or co-culturing ADSCs and cells with secretion functions to promote differentiation in the nerve direction, researchers also adopt a gene transfection technology to transfer BDNF and neurotrophic factor-3 (neurotropin-3) into adipose mesenchymal stem cells of SD rats through lentiviruses and induce the cells through the nerve culture medium, so that the amount of expressed NSE genes is increased.

CN105567637A discloses that human adipose-derived stem cells are induced and differentiated into motor neuron cells by adopting SHH and RA together, and the obtained motor neuron cells are characterized by specific nerve cell marker protein; the specific nerve cell marker proteins are HB9 and Oligo 2; and simultaneously, identifying the obtained motor neuron cells by utilizing the electrophysiological function.

However, although the induction mode of the adipose-derived mesenchymal stem cells towards the nerve direction is various and the method is numerous, there is still room for further improvement.

Disclosure of Invention

The invention overcomes the defects of the prior art and provides an improved method for promoting the differentiation of adipose-derived mesenchymal stem cells into nerve cells.

Furthermore, the invention provides a method for identifying differential genes, wherein two genes with larger difference are obtained by screening through analyzing the expression difference of adipose-derived mesenchymal stem cells before and after induction to neural cells, and the two genes are presumed to have the function of promoting differentiation or proliferation of the stem cells to the neural cells.

The invention also provides a method for separating the adipose-derived mesenchymal stem cells.

Specifically, the separation method includes a separation method which is conventional in the art.

Further, the method for isolated culture of adipose-derived mesenchymal stem cells comprises the steps of taking about 300mg of adipose tissue, repeatedly washing with PBS containing 200U/mL penicillin and 200mg/L streptomycin, shearing the adipose tissue into paste in the ophthalmology, transferring the paste into a culture bottle, adding 0.1% collagenase I with 3 times of volume, oscillating and digesting the paste in a constant-temperature water bath kettle at 37 ℃ for 70min, wherein the liquid level is divided into 3 layers, the upper layer is a yellow oily adipose cell layer, the middle layer is an adipose tissue layer, the lower layer is a collagenase layer containing mononuclear cells, centrifuging the mixture at 1500r/min for 15min, discarding the supernatant, completely adding a complete culture solution (containing 10% fetal calf serum by volume and 100 × double antibody HG-DMEM culture medium), blowing and precipitating the cell, filtering the cell by a 200-mesh cell sieve, and adding 5% CO with volume fraction at 37 ℃ to 5%₂Culturing under saturated humidity condition. After 24h, the liquid is changed for the first time, when 80% of the liquid is fused after growth, trypsin and EDTA mixed with 1:1 are used for digestion and passage, and the obtained adipose-derived mesenchymal stem cells are subjected to continuous passage culture in vitro.

Furthermore, the invention provides an application of the NUDT12 gene in promoting differentiation of adipose-derived mesenchymal stem cells into nerve cells.

Further, a method for inducing adipose-derived mesenchymal stem cells to differentiate into neural cells is provided, specifically, a human neural inducing solution is added to the adipose-derived mesenchymal stem cells: after 3 days, the nerve induction agents 120umol/L indomethacin, 3mg/L insulin and 300umo1/LIBMX are added into the mixture of 10ug/LEGF, 20ug/LbFGF and 10 ug/LBDNF.

The invention also provides a transgenic adipose-derived mesenchymal stem cell.

Specifically, the preparation method of the transgenic adipose-derived mesenchymal stem cell comprises the step of designing an upstream primer and a downstream primer according to the sequence of the NUDT12 gene. Extracting adipose-derived mesenchymal stem cell RNA by a TRIzol method, and performing reverse transcription to generate cDNA; and (3) performing PCR amplification by using the cDNA as a template. And carrying out agarose gel electrophoresis on the gene product obtained after PCR amplification, and recovering the target fragment by using a gel recovery kit. And (3) carrying out double enzyme digestion on the target fragment and the pLVX-IRES-ZsGreenl empty vector respectively, and recovering an enzyme digestion product by using a DNA product purification kit. Mixing the recovered target fragment with a vector, and connecting the target fragment and the vector by using DNA ligase overnight; then, the system is used for transforming Top10 escherichia coli, plasmids are extracted and identified through enzyme digestion, and the correctly identified recombinant plasmids are used for cell transfection. And (3) slow virus packaging: 293FT cells were seeded into 5cm plates in advance in 5ml DMEM medium containing 10% FBS, 5% CO₂Culturing at 37 deg.C, transfecting when cell confluence reaches about 80%, replacing culture solution about 12h after co-transfecting the prepared recombinant lentivirus plasmids 5ug, pCMV-VSVG7ug and pCMV △ 8.917ug, culturing for 72h, collecting cell supernatant rich in lentivirus, removing cells with 0.45 μm filter membrane, concentrating with concentration kit, and concentrating BMSCs with good growth and uniform morphology at cell density of 1x10⁵Perwell were inoculated into 24-well plates. 37 ℃ and 5% CO₂Incubating in incubator overnight, infecting BMSCs with the synthesized lentivirus at MOI of 100 for 2h, supplementing 250uL of normal medium containing 10% FBS in 5% CO₂Culturing in an incubator; after the virus infects the cells for 12h, the culture medium is discarded, and 500uLIMDM culture medium is added into each well. After the virus is infected for 96h, positive transgenic cells are screened and cultured for later use.

Further, a method for inducing the transgenic adipose-derived mesenchymal stem cells to differentiate into nerve cells is provided, and specifically, a human nerve inducing solution is added to the transgenic adipose-derived mesenchymal stem cells: l0ug/LEGF, 20ug/LbFGF and 10ug/LBDNF, after 3 days, adding 120umol/L indomethacin, 3mg/L insulin and 300umo1/LIBMX as nerve inducing agent.

A nerve cell is prepared by the method for inducing the differentiation of the adipose-derived mesenchymal stem cells.

The invention has the advantages of

According to the invention, two genes with large difference are obtained by screening through analyzing the expression difference of the adipose-derived mesenchymal stem cells before and after induction to neural cells, and the two genes are presumed to have the effect of promoting differentiation or proliferation of the stem cells to the neural cells. After the NUDT12 gene is over-expressed in the adipose-derived stem cells, the induction efficiency of nerve cells and the proliferation speed of the induced nerve cells are remarkably improved, and the nerve cell identification has a good effect.

Drawings

FIG. 1 cell proliferation Activity graph

FIG. 2 is a detection chart of Western blotting

FIG. 3 is a graph showing the results of whole-cell patch clamp on the resting potential of cells

The specific implementation mode is as follows:

the present invention is described in detail below by way of examples, it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and those skilled in the art can make some insubstantial modifications and adaptations of the present invention based on the above-described disclosure.

Example 1 isolated culture of adipose-derived mesenchymal stem cells

Taking about 300mg of adipose tissue, repeatedly cleaning with PBS containing 200U/mL penicillin and 200mg/L streptomycin, shearing to paste by ophthalmic scissors, transferring into a culture bottle, adding 0.1% type I collagenase with 3 times volume, and performing oscillatory digestion in a constant temperature water bath kettle at 37 ℃ for 70min, wherein the liquid level is divided into 3 layers, the upper layer is a yellow oily adipose cell layer, the middle layer is an adipose tissue layer, and the lower layer is a collagenase layer containing mononuclear cells. 1500 r-Centrifuging for 15min, discarding supernatant, adding complete culture solution (containing 10% fetal calf serum and 100 × double antibody HG-DMEM culture medium) to blow and beat cell sediment, sieving and filtering with 200 mesh cell sieve, culturing at 37 deg.C and 5% CO2 saturated humidity for 24h, first replacing liquid, when the cell grows to 80% fusion, digesting and passaging with 1:1 mixed trypsin and EDTA, culturing the obtained adipose mesenchymal stem cells in vitro by continuous passage, taking the 4 th generation adipose mesenchymal stem cells with good growth state, washing with PBS for 4 times (centrifuging at 1500r/min for 15min), adjusting cell density to 1 ×/L, absorbing fluorescence labeled monoclonal antibody 5 μ L (mouse anti-human CD13-FITC, CD34-PE, CD 2-FITC, CD59-PE, HLA-DR-FITC labeled monoclonal antibody), adding suspension 100 μ L, mixing, adjusting PBS for 15min, adding 500 μ L, incubating, and performing a flow-type test on the surface of the cells to obtain CD 94. 13% positive expression rate of adipose cells, and detecting the result of the cells by using a CD-DR-FITC test instrument, wherein the surface of the CD 3% of the cells shows that the positive expression rate of the adipose-CD 3 is equal to 10.35%, the positive expression of the following adipose-CD 3, the negative cell suspension of the adipose-CD 3, the adipose-CD 3 cell suspension is obtained by adding the test instrument, the test instrument shows that the test instrument that the adipose-CD 3 instrument shows that the adipose-⁷and/L is reserved.

Example 2 Induction experiment of adipose-derived mesenchymal Stem cells into neural cells

When the 3 rd generation adipose-derived stem cells are 80% full, the complete culture medium is discarded, the complete culture medium is continuously added into a control group, and the induction method adopted by the experimental group is as follows: adding human nerve induction liquid: l0ug/L EGF, 20ug/L bFGF and 10ug/LBDNF, 3d later neural inducer: 120umol/L indometacin, 3mg/L insulin, 300umo 1/LIBMX. After adding the nerve inducer for 48h, differentiation of the ADSCs into nerve cells was observed, the liquid in the 6-well plate was discarded, and the plates were washed 2 times with PBS for 5min each time. Add the same permeabilizing reagent (methanol: acetone ═ 1: 1), wash with PBS 3 times after 30min, 5min each time. Then anti-GFAP primary antibody (diluted 1: 50), anti-beta-tubulin III primary antibody (diluted 1: 50) were added, an equal amount of PBS was added to the control group, incubation was performed overnight at 4 ℃, goat anti-human FITC secondary antibody (diluted 1: 100) was added, incubation was performed lh in the dark at 37 ℃ and washing was performed 3 times with PBS. Finally, DAPI staining solution was added, and the mixture was washed with PBS for 5min at room temperature for 3 times. And (4) observing under a fluorescence microscope. After immunofluorescent staining of the sample, 5 different fields of view were observed at random under a low power microscope, the number of positive GFAP and beta-tubulin III cells in 50 cells in each field of view was counted, and the results were analyzed by an image analysis system and are shown in Table 1.

TABLE 1 GFAP,. beta. -tubulin III positive cell rate (%)

	GFAP	β-tubulin III
			Nerve cell induction	73.27±5.1	66.29±4.3

Positive cells were selected for use.

Example 3 bioinformatic analysis screening of neural cell-associated genes

The applicant carries out expression profile analysis on cells obtained before differentiation of human adipose-derived mesenchymal stem cells into nerve cells and after induction in example 2, firstly obtains an expression value of an original gene, analyzes data through R software, analyzes the final data by using a limma function package to obtain a differential expression gene before and after differentiation, and determines differential analysis conditions that the expression multiple of the gene is more than 1 time and the FDR jadeite is 0.01. 2 genes with the most significant differential expression induced by the differentiation regulators are obtained by final screening through analysis, and are specifically shown in table 1.

TABLE 1 differentially expressed genes

Gene	Fold change
		Homosapiensnudixhydrolase12(NUDT12)	3.271
Homo sapiens syntaxin 1B(STX1B)	4.023

Based on the changes of the two different genes, it was preliminarily determined that the two genes should have positive promoting effects on the proliferation and induced differentiation of nerve cells.

Example 4 transfection of human adipose mesenchymal Stem cells by NUDT12

Based on the sequence NM-031438.4 of NUDT12 gene, upstream and downstream primers were designed. Are respectively

An upstream primer: 5'-aaaactcgagaagactgcatccggctccag-3', respectively;

a downstream primer: 5'-aaaagcggccgctagattttaaaaatttaatgt-3' are provided.

Extracting adipose-derived mesenchymal stem cell RNA by a TRIzol method, and performing reverse transcription to generate cDNA; using cDNA as template. And (3) PCR reaction conditions:

5min at 98 ℃; 15S at 98 ℃, 35S at 65 ℃ and 3min at 72 ℃ for 30 cycles; extension at 72 ℃ for 10 min. And carrying out agarose gel electrophoresis on the gene product obtained after PCR amplification, and recovering the target fragment by using a gel recovery kit. And (3) carrying out double enzyme digestion on the target fragment and the pLVX-IRES-ZsGreenl empty vector respectively, and recovering an enzyme digestion product by using a DNA product purification kit. Recovering the target fragment and the vector in a ratio of 6: 1, and ligating overnight at 4 ℃ with DNA ligase; then, the system is used for transforming Top10 escherichia coli, clones are screened on an LB agar plate selected by ampicillin resistance, positive clones are selected, LB is shaken for a while, and plasmids are extracted by a small extraction kit; and enzyme digestion identification is carried out, and the correctly identified recombinant plasmid is used for cell transfection.

And (3) slow virus packaging: 293FT cells were seeded into 5cm plates in advance in 5ml DMEM medium containing 10% FBS, 5% CO₂Culturing at 37 deg.C, transfecting when the cell confluence reaches about 80%, replacing culture solution about 12h after co-transfecting the prepared recombinant lentivirus plasmids 5ug, pCMV-VSVG7ug and pCMV △ 8.917ug, continuously culturing for 72h, collecting cell supernatant rich in lentivirus, removing cells with 0.45 μm filter membrane, concentrating by using concentration kit, determining virus titer by genome Q-PCR, determining virus titer as follows, pLVX-IRES-ZsGreenl-NUDT 12, determining the virus titer is 1.57X10¹¹TU/ml。

The cell density of the P3 generation BMSCs with good growth and uniform morphology is 1x10⁵Perwell were inoculated into 24-well plates. 37 ℃ and 5% CO₂Incubating in incubator overnight, infecting BMSCs with the synthesized lentivirus at MOI of 100 for 2h, supplementing 250uL of normal medium containing 10% FBS in 5% CO₂Culturing in an incubator; after the virus infects the cells for 12h, the culture medium is discarded, and 500uLIMDM culture medium is added into each well. After the virus is infected for 96 hours, the transfected cells are observed under a fluorescence microscope, the infection efficiency of the cells is analyzed to be 76.9 percent by photographing, and positive transgenic cells are screened and cultured for later use.

Example 5 Induction experiment of transgenic adipose-derived mesenchymal Stem cells into neural cells

When the transgenic adipose-derived stem cells prepared in example 3 and the control 3 rd generation adipose-derived stem cells are respectively cultured until 80% of the cells are fully paved, the complete culture medium is discarded, and a human nerve induction solution is added: l0ug/L EGF, 20ug/L bFGF and 10ug/LBDNF, 3d later neural inducer: 120umol/L indometacin, 3mg/L insulin, 300umo 1/LIBMX. After adding the nerve inducer for 48h, the liquid in the 6-well plate was discarded, and the plate was washed 2 times with PBS for 5min each time. Add the same permeabilizing reagent (methanol: acetone ═ 1: 1), wash with PBS 3 times after 30min, 5min each time. Then anti-GFAP primary antibody (diluted 1: 50), anti-beta-tubulin III primary antibody (diluted 1: 50) were added, an equal amount of PBS was added to the control group, incubation was performed overnight at 4 ℃, goat anti-human FITC secondary antibody (diluted 1: 100) was added, incubation was performed lh in the dark at 37 ℃ and washing was performed 3 times with PBS. Finally, DAPI staining solution was added, and the mixture was washed with PBS for 5min at room temperature for 3 times. And (4) observing under a fluorescence microscope. After immunofluorescent staining of the sample, 5 different fields of view were observed at random under a low power microscope, the number of positive GFAP and beta-tubulin III cells in 50 cells in each field of view was counted, and the results were analyzed by an image analysis system and are shown in Table 1.

TABLE 1 GFAP,. beta. -tubulin III positive cell rate (%)

From the results, the transgenic adipose-derived mesenchymal stem cells have significant GFAP and beta-tubulin III positive cell rate compared with non-transgenic adipose-derived mesenchymal stem cells, which fully indicates that the transgenic adipose-derived mesenchymal stem cells have better differentiation effect towards nerve cells.

Example 6 detection of proliferating Activity of neural cells after differentiation by MTT method

1. After the neural cells obtained by differentiation in example 4 were cultured for passage 3 (as a control, neural cells obtained by inducing non-transgenic adipose-derived stem mesenchymal stem cells by the same induction method), 0.25% trypsin was digested, the medium was counted after resuspension, the cell suspension was diluted, and the concentration was adjusted to 104/ml per well;

2. inoculating 150 μ l of cells to a 96-well plate, and repeating 5 parallel wells;

3. after 1-6 days of transfection, the medium in each well was discarded, and 100. mu.l (0.5mg/ml) of MTT medium was added to continue culturing for 5 hours. Discarding the MTT culture solution, adding 150 mul DMSO into each well to dissolve MTT reducing substance, shaking on a shaking table for 10min to fully dissolve the crystal, and detecting the absorbance value at 490nm by an enzyme linked immunosorbent assay;

4. the cellular absorbance values were counted every day and the resulting values were plotted in a graph.

5. Results

The result is shown in fig. 1, compared with the control, the proliferation rate of the nerve cells in the transgenic group is obviously increased, which suggests that increasing the expression level of the NUDT12 gene can change the proliferation capacity of the differentiated nerve cells, and has better promoting effect.

Example 7Western blotting detection of overexpression of NUDT12

Collecting the transgenic adipose-derived stem cell induced in example 4, removing the culture medium, washing with pre-cooled PBS for 2 times, adding pre-cooled cell lysate RIPA containing protease inhibitor PMSF, scraping the cell into a centrifuge tube after ice bath for 5-10 min, performing ultrasonic treatment on ice for 2min, centrifuging at 12000 Xg, collecting the supernatant, quantifying the protein concentration with a spectrophotometer, subjecting 8ug of the protein sample to 10% SDS-PAGE electrophoresis and wet transfer to PVDF membrane, sealing the PVDF membrane with 5% skimmed milk powder for 1h, incubating overnight at 4 ℃ with rabbit anti-NUDT 12 monoclonal antibody (ab197310) (1:1000), washing the membrane with TBST for 3 times, incubating with goat anti-rabbit secondary antibody (ab205718) (1:5000) for 1h, washing the membrane with TBST for 3 times, performing ECL chemiluminescence coloration, scanning with Tanon 4600 full-automatic chemiluminescence image analysis system, and analyzing the result, as shown in FIG. 2, the NUDT12 protein is highly expressed in the transgenic neural cell relative to the actin protein, the expression level is increased by about 425%, and the method has a good improvement effect.

Example 8 functional assay of neuronal cells following induction of MSCs

Detecting cell resting potential by using whole-cell patch clamp: the differentiated nerve cell group is selected from nerve-like cells with strong refractivity, two or more long bulges and round cell bodies for experiments. The external diameter of the microelectrode is 1.2mm, the internal diameter is 0.5mm, and the liquid in the microelectrode is flushed and filled. The resistance is 2-5M, the sampling frequency is 10kHz, and the low-pass filtering is 1 kHz. And adopting a high-impedance sealing technology and punching under negative pressure to form cell clamping. Whole cell recordings were performed using the AXOPATCH 200B amplifier protocol. The resting membrane potential of the adipose derived mesenchymal stem cells in the control group is-11.57 +/-2.35 mV, and the resting membrane potential of the induced nerve cells is obviously increased to-29.32 +/-4.87 mV (p is less than 0.05), which indicates that the nerve cells have electrophysiological functions. The nerve cells obtained after the transgenic adipose-derived stem cells are induced have more negative membrane potential 35.88 + -3.02 (p <0.05) than the non-transgenic induced neuron-like cells (see figure 3). The result proves that the nerve cells obtained after the induction of the transgenic adipose-derived mesenchymal stem cells have more advantages in function.

It is to be understood that the invention is not necessarily limited in its application to the details of construction and the arrangement of components set forth in the following description and/or illustrated in the drawings. The invention is capable of embodiments in addition to those described and of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein, as well as the abstract, are for the purpose of description and should not be regarded as limiting.

As such, those skilled in the art will appreciate that the conception, upon which this disclosure is based, may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.

Claims

Use of the NUDT12 gene in promoting differentiation of human adipose-derived mesenchymal stem cells into nerve cells.
Use of overexpression of NUDT12 in promoting differentiation of human adipose-derived mesenchymal stem cells into neural cells and promoting rapid proliferation of the differentiated neural cells.
3. A method for inducing human adipose-derived mesenchymal stem cells to differentiate into nerve cells and promoting the rapid proliferation of the differentiated nerve cells is characterized by comprising the steps of constructing adipose-derived mesenchymal stem cells with transformed NUDT12 genes, and performing induced differentiation and cell culture on the transgenic adipose-derived mesenchymal stem cells.
4. The neural cell produced by the method according to claim 3, which is produced by the above method for inducing neural differentiation of a stem cell.
5. The method of claim 3, wherein: the specific induction method comprises the following steps: adding human nerve induction liquid to adipose mesenchymal stem cells: l0ug/L EGF, 20ug/L bFGF and 10ug/L BDNF, 3d later a neuro-inducer was added: 120umol/L indometacin, 3mg/L insulin, 300umo 1/LIBMX.
6. The method according to claim 3, wherein the step of constructing the adipose-derived mesenchymal stem cell transformed with the NUDT12 gene comprises designing an upstream primer and a downstream primer according to the sequence of the NUDT12 gene. Extracting adipose-derived mesenchymal stem cell RNA by a TRIzol method, and performing reverse transcription to generate cDNA; and (3) performing PCR amplification by using the cDNA as a template. And carrying out agarose gel electrophoresis on the gene product obtained after PCR amplification, and recovering the target fragment by using a gel recovery kit. And (3) carrying out double enzyme digestion on the target fragment and the pLVX-IRES-ZsGreenl empty vector respectively, and recovering an enzyme digestion product by using a DNA product purification kit. Mixing the recovered target fragment with a vector, and connecting the target fragment and the vector by using DNA ligase overnight; then, the system is used for transforming Top10 escherichia coli, plasmids are extracted and identified through enzyme digestion, and the correctly identified recombinant plasmids are used for cell transfection. And (3) slow virus packaging: 293FT cells were seeded into 5cm plates in advance in 5ml DMEM medium containing 10% FBS, 5% CO₂Culturing at 37 deg.C, transfecting when cell confluence reaches 80%, co-transfecting the prepared recombinant lentivirus plasmids 5ug, pCMV-VSVG7ug and pCMV △ 8.917ug for about 12h, culturing for 72h, collecting cell supernatant rich in lentivirus, removing cells with 0.45 μm filter membrane, concentrating with concentration kit, and concentrating BMSCs with good growth and uniform morphology at cell density of 1x10⁵Perwell were inoculated into 24-well plates. 37 ℃ and 5% CO₂Incubating in an incubator overnight, infecting adipose tissue-derived mesenchymal stem cells with the synthesized lentivirus at an MOI value of 100, supplementing 250uL of a normal medium containing 10% FBS (fetal bovine serum) after 2h, and adding 5% CO₂Culturing in an incubator; after the virus infects the cells for 12h, the culture medium is discarded, and 500uLIMDM culture medium is added into each well. After the virus is infected for 96h, positive transgenic cells are screened and cultured for later use.