Screening Of Small Molecules Of Traditional Chinese Medicine Regulating SOX9 Gene And Preliminary Study On Its Effect On Chondrocyte Phenotype

Objective: To screen out the small molecule of Chinese medicine that can regulate SOX9 gene and explore its effect on chondrocyte phenotype.Methods: 1.Recombinant plasmid p GL3-SOX9 was constructed and identified by gene sequencing and nucleic acid electrophoresis.2.The established model was used to conduct primary screening and re-screening of the small molecule library of 308 compounds to obtain the small molecule of Chinese medicine that could promote the expression of SOX9 gene;3.The cartilage cell separation and identification,by trypsin and collagenase digestion,Ⅱ type separation for the pig cartilage cells,in vitro after a period of time to observe the morphology of cartilage cells under a microscope.4.Traditional Chinese medicine(TCM)effect of small molecules on chondrocyte phenotype with CCK8 method determination of different concentrations of traditional Chinese medicine(TCM)small molecules to the influence of the cartilage cell proliferation activity,set up the blank control group(DMSO),Chinese medicine small molecular groups to join the medium respectively,and then using PCR,Western blot and immunofluorescence technique of SOX9 and downstream into cartilage test the different expression of related genes.Results:1.Successful build p GL3-SOX9 recombinant plasmid,and apply gene sequencing and DNA electrophoresis confirmed the extracted plasmid is p GL3-SOX9 recombinant plasmid,and small molecules out of seven kinds of Chinese medicine,113,114,78,72,118,129 and 672 were in a certain extent promote the SOX9 gene expression,the no.72 and no.129 small molecules is relatively significant two kinds of traditional Chinese medicine.2.CCK8 method was used to determine that in the range of 5 μg/m L-40 μg/ml,different concentrations of TCM small molecules 72 and 129 had no obvious adverse effects on the growth activity of chondrocytes.By PCR,Western blot,immunofluorescence detection technology,and confirmed the number 129,72 Chinese medicine small molecules of SOX9 and COL-Ⅱ gene expression,compared with blank control group(DMSO)were improved,the 129 th Chinese medicine small molecule is more obvious.Conclusion:1.The screening model of SOX9 gene expression regulator was constructed,and the TCM small molecules that promoted the activity of SOX9 promoter were successfully screened out from308 kinds of TCM small molecules(No.72 and No.129).2.TCM small molecule No.72 and No.129 can maintain the phenotype of chondrocytes by promoting the expression of SOX9 gene,among which No.129 is more significant.