orc1基因rna干扰对大鼠血管平滑肌细胞表型影响 - 第三军医大学学报.docVIP

ORC1基因RNA干扰对大鼠血管平滑肌细胞表型影响 李建涛，舒茂琴1▲，冯媛媛（第三军医大学西南医院心血管内科，重庆市介入心脏病学研究所，重庆，400038） 摘要：目的　以RNA干扰抑制血管平滑肌细胞( vascular smooth muscle cells, VSMCs)ORC1基因,探讨ORC1基因表达抑制后VSMCs表型的改变。方法　实验设置正常对照组、阴性siRNA组及阳性siRNA组。应用Western blot检测ORC1基因表达的变化；免疫荧光染色后激光共聚焦显微镜观察细胞形态结构；应用流式细胞仪检测细胞表型标志蛋白表达。结果　①siRNA转染后，阳性siRNA组ORC1基因表达水显著降低，而正常对照组及阴性siRNA组间ORC1基因表达水平无显著差异。②siRNA转染后，阳性转染组细胞呈现收缩型形态特征，空白对照组及阴性对照组细胞呈现合成型形态特征 ③siRNA转染使ORC1表达减弱后，VSMCs收缩型标志物α平滑肌肌动蛋白（α-SMactin）和平滑肌肌动蛋白重链2（SM-2）表达水平明显升高，合成型标志物骨桥蛋白（Osteopontin）RNA干扰介导的ORC1基因沉寂可促使VSMCs由合成型向收缩型转变。 关键词: ORC1； RNA干扰；血管平滑肌细胞；表型 Effect of RNA interference for ORC1 gene on phenotype of vascular smooth muscle cells in rats LI Jian-Tao，SHU Mao-qin，(Department of Cardiology，Southwest Hospital, Third Military Medical University, Chongqing 400038, China) Abstrct: Objective To investigate the influence of RNA interference targeting ORC1 gene on the phenotype of vascular smooth muscle cells(VSMCs) in rat. Methods VSMCs were transfected with siRNA targeting ORC1 gene by liposome. The expression of ORC1 protein was detected by Western blotting. The changes of phenotype-dependent markers was analyzed by flow cytometry. Results After transfected of the pair of siRNA targeting ORC1 genes, the expression of ORC1 was lower than that in the control group ( non transfection and negative siRNA). ORC1 gene silencing evoked drastic changes in characteristics of the VSMC de-differentiated phenotype, including VSMC morphology and the expression of phenotype-dependent markers in favor of differentiation. Conclusion ORC1 gene silenced by RNA intereference can mediate the transition of VSMCs from synthetic phenotype to contractile phenotype. Key words: ORC1; RNA interference; vascular smooth muscle cell; phenotype 　 中图法分类号: R322.12； R329.28； R394.2　　　文献标识码: A 血管平滑肌细胞( vascular smooth muscle cells,VSMCs)的增殖是动脉粥样硬化（Atherosclerosis As）[1]，抑制VSMCs的增殖并使其维持在分化状态在是防治这类病理生理过程发生、发展从而减轻血管重构的重要环节。发育成熟的正常动脉组织中，VSMCs 处于分化状态，具有收缩能力，形态呈纺锤形并表达与收缩功能相关的标志蛋白。血管损伤后，位于中膜的VSMCs 增殖转化为去分化表型