背景：目前有研究表明，很多细胞生长因子可促进骨骺干细胞的增殖和分化，并且这种调控作用与Sox-9有密切的关系。 目的：观察小分子干扰RNA沉默Sox-9基因及对大鼠骨骺干细胞增殖、凋亡的影响。 方法：采用酶消化法分离、免疫磁珠法纯化及免疫组织化学SABC法鉴定得到大鼠骨骺干细胞，首先进行预转染Sox-9 siRNA，判断转染效率，实验分2组：对照组常规培养，实验组采用Sox-9 siRNA转染。 结果与结论：实验成功分离、纯化了原代骨骺干细胞，生长状态稳定、贴壁牢固，光学显微镜下细胞多呈梭形。免疫组织化学染色和免疫荧光鉴定显示，Sox-9 siRNA转染的大鼠骨骺干细胞可稳定表达相对特异性标志物成纤维细胞生长因子受体3。RT-PCR，MTT，流式细胞仪检测检结果提示，与对照组相比，实验组Sox-9 mRNA表达及细胞存活率降低(P<0.05)，凋亡率增加(P<0.05)。结果证实，siRNA沉默Sox-9基因可调控大鼠骨骺干细胞的增殖和凋亡。%BACKGROUND:Current research has shown that many cellgrowth factors can promote the proliferation and differentiation of epiphysis stem cells, and this regulation is closely related to Sox-9. OBJECTIVE:To investigate the effects of smal interfering RNA-induced specific silence of Sox-9 gene on the proliferation and apoptosis of epiphysis stem cells. METHODS:Epiphysis stem cells were isolated from the ring of La Croix with enzyme digestion and purified with magnetic activated cellsorting, identified by immunocytochemistry assay. The cells in good conditions were selected to be transfected by Sox-9 silenced by smal interfering RNA with fluorescent marker, and then were observed under the fluorescent microscope to check the transfection efficiency. Next step, epiphysis stem cells were divided into 2 groups:group one was cultured normal y as control group;group two was transfected by Sox-9 silenced by smal interfering RNA through LipofectamineTM 2000 as experimental group. RESULTS AND CONCLUSION:The primary epiphysis stem cells were separated and purified, which were of stable growth and tightly attachment. The results of immunohistochemistry and immunofluorescence showed the epiphysis stem cells expressed cel-specific markers, fibroblast growth factor receptor 3. After transfection, reverse transcription-PCR results showed that Sox-9 gene expression in epiphysis stem cellwas inhibited specifical y;3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide results showed that cellproliferation in experimental group decreased significantly compared with the control group (P<0.05), and the cellapoptosis detected by flow cytometry showed that the experimental group increased as compared with the control group (P<0.05). Sox-9 gene plays an important role in regulating the proliferation and apoptosis of rat epiphysis stem cells.
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