Snake Venom Nerve Growth Factor Regulate Autophagy Inducing Chondrogenic Differentiation Of BMSCs And Its Mechanism

OBJECTIVE: In recent years,more and more attention has been paid to the role of autophagy in cells.This study is to investigate the role of Guangxi cobra venom never growth factor(NGF)in regulating the chondrogenic differentiation of stem cells through the autophagy,and provide an experimental method for further exploring the molecular biological mechanisms of NGF induced stem cell chondrogenic differentiation.METHODS: The experiment were divided into four groups:(1)blank control group,(2)autophagy induction group,(3)NGF group,(4)TGF-? group.The following indicators were test after induction of stem cells in vitro on day 3,5 and 7 :(1)Cell survival assay: FDA/PI staining,DNA content detection;(2)Chondrogenic differentiation assay: Safranin O staining,HE Staining,glycosaminoglycan(GAG)content detection,immunofluorescence(type I collagen,type II collagen),RT-PCR(COL1A1,COL2A1,SOX9,ACAN);(3)autophagy-related detection: Stub-RFP-Sens-GFP-LC3 lentivirus transfection monitored autophagic flow;transmission electron microscopy(TEM);Western Blot(LC3I/II,ATG5,ATG7,BECN-1,P62).Then the genes related to autophagy were screened out by chip technology.Specific experiments were as follows: After RNA sequencing was completed,the genes with differentially expressed genes were screened from the large volume data.Through the bioinformatics technique analyz the differential gene.The expression of related genes was verified by RT-PCR,and consulted the relevant literature selected significance and innovation Genes.The target gene were silenced by means of plasmid transfection.The efficiency of transfection was verified by RT-PCR at the gene level.After successful transfection,normal stem cells and stem cells of gene-silenced were cultured,conducted DNA content determination,FDA/PI staining,HE staining,GAG content detection,immunofluorescence staining,RT-PCR,and Stub-RFP-Sens-GFP-LC3 lentivirus transfection,TEM,Western Blot methods to further observe the effects of the target gene on the chondrogenic differentiation of stem cells by NGF.RESULTS: The effects of NGF on inducing chondrogenic differentiation of stem cells in vitro were as follows:(1)FDA/PI staining and DNA content detection showed that the number of cells increased with increasing induction time;(2)Detection of cartilage differentiation markers: Safranin O,HE,GAG content,immunofluorescence(type I collagen,type II collagen),RT-PCR(COL1A1,COL2A1,SOX9,ACAN),the results showed that chondrocytes specific characteristics appeared in the NGF and TGF-? groups,which can secrete chondrocyte extracellular matrix(glycosaminoglycan and chondroitin sulfate),while cartilage-specific genes COL2A1,SOX9,ACAN are significantly upregulated;(3)Autophagy-related detection: RFP-GFP-LC3 double Fluorescence lentivirus assay;TEM results showed that more autophagosomes can be seen in the NGF group than in the control group.Western Blot(LC3I/II,ATG5,ATG7,BECN-1,P62)results showed that NGF can be significantly upregulates key autophagy proteins.Based on gene chip technology,through some bioinformatics methods,differential expression of genes were analysis volcanic map,KEGG Pathway function enrichment.Screening and analysis genes associated with autophagy for heat map analysis,drawing KEGG Pathway pathway maps and protein interaction network diagram.More intuitive relationship between differential gene and stem cell differentiation associated with autophagy.The results of RT-PCR validation showed that the expression of autophagy-related genes was significantly up-regulated in the NGF group,indicating that autophagy occurred during the differentiation of stem cells into cartilage.The target genes were for plasmid transfection.After 8 days of culture,the green fluorescence was significantly expressed.The expression of the target genes were detected by RT-PCR.The experimental results showed that the expression level of the target genes were significantly higher in the normal group than in the gene silencing group(P<0.05),gene silencing was successful.In vitro study of NGF induced chondrocyte differentiation,cultured normal stem cells and gene-silenced cells for 7 days,normal stem cells under the action of NGF inducing cartilage-related genes COL2A1,SOX9,ACAN expression and gene silencing group There was a significant difference(P<0.05).Meanwhile,the results of RT-PCR were further confirmed by DNA content determination,HE staining,FDA/PI staining,and GAG content determination.In autophagy-related assays,the expression level of autophagy in the NGF group was significantly higher than that in the gene silencing group.CONCLUSION: The above experimental results indicated that NGF can induce chondrogenic differentiation of stem cells in vitro.Based on gene chip technology,selected target genes were silenced by plasmid transfection,further confirming that NGF played a role in inducing chondrogenic differentiation of stem cells through autophagy in vitro.The results of this experiment provide the experimental basis for exploring molecular biology mechanisms of NGF on chondrogenic differentiation of stem cells,and will be better applied to cartilage repair.