Experimental Study On The Effects Of Sirna Silencing Sox-9 Gene On The Biological Characteristics Of Epiphysis Stem Cell

Objective This study was conducted to discuss the effects of Sox-9 gene silencing by siRNA interference on the cell proliferation, apoptosis, chondrocyte differentiation and other biological characteristics of epiphysis stem cell. To learn about the related molecular mechanism of the regulation by Sox-9 gene on PTHrP, the cartilage extracellular matrix as Col2α1, aggrecan of epiphysis stem cell.Method Assisted with the microscopic surgical techniques, epiphysis stem cells were isolated with enzyme digestion from the ring of La Croix, which is in the neonatal rat's metaphysics and purified with magnetic activated cell sorting, identified by immunocytochemistry and immunofluorescence with the specific marker (FGFR-3). At first, the cells in good conditions were selected to be transfected by Sox-9 siRNA with fluorescent marker, and then were observed under the fluorescent microscope to check the transfection efficiency and get the optimal transfection conditions. Next step, epiphysis stem cells were divided into 2 groups: one was cultured normally as the control group; the other was transfected by Sox-9 siRNA through Lipofectamine 2000 as experimental group. The cells were detected with MTT to assess cell proliferation at 24h, 48h, 72h three time points and flow cytometry to assess cell apoptosis at 48h. The cells were collected for RT-PCR detection of Sox-9, Col2α1, aggrecan, PTHrP, Ihh gene expression at 48h. Sox-9 siRNA transfection efficiency and proliferation, apoptosis and chondrocyte differentiation of the epiphysis stem cell after interference were evaluated according to the results.Result 1. The primary epiphysis stem cells were separated and purified, which were of stable growth and tightly attachment. 2. The results of Immunohistochemistry and immunofluorescence showed the epiphysis stem cells express cell-specific markers FGFR-3; 3. Pre-experiments before transfection showed that all the transfection efficiency of transfection group were more than 80%, which indicated the follow-up experiment could be processed. 4. MTT results showed that cell proliferation in experimental group decreased contrast with the control group (p < 0.05); 5. The cell apoptosis detected by flow cytometry showed that the experimental group increased contrast with the control group; 6. RT-PCR results showed that Sox-9, Col2α1, aggrecan, PTHrP gene expression of the experimental group were lower than the control group, but Ihh expression was not changed obviously.Conclusion The Sox-9 gene expression of epiphysis stem cells could be interfered efficiently by siRNA transfection. The cell proliferation could be inhibited while the cell apoptosis were not be affected obviously after the silence of Sox-9 gene by the siRNA interference. The cartilage extracellular matrix as Col2α1 and aggrecan gene expression of epiphysis stem cell was inhibited, and the expression of PTHrP gene also reduced, but the expression of Ihh gene was not changed obviously, which indicated that the inhibitory effect of the cell proliferation of epiphysis stem cells by Sox-9 siRNA transfection maybe through the inhibiting the expression of PTHrP gene. The above results show that Sox-9 gene plays an important role in the regulation of epiphysis stem cells, and makes the epiphysis stem cells as seed cells for cartilage tissue engineering possible in the future.