microRNA在高磷诱导血管平滑肌细胞钙化早期的动态变化  

Dynamic alteration of microRNA in high phosphorus induced calcification of vascular smooth muscle cell

作　　者：肖洋[1] 杜瑶瑶[1] 高成[1] 孔炜[1] 

机构地区：[1]北京大学基础医学院生理学与病理生理学系,教育部分子心血管学重点实验室,北京100191

出　　处：《北京大学学报（医学版）》2016年第5期756-765,共10页Journal of Peking University:Health Sciences

基　　金：国家基金委重大国际合作项目(81220108004);国家重点基础研究发展计划(973计划,2012CB518002);国家自然科学基金(81070243,81121061,91339000);国家杰出青年基金(81225002)资助~~

摘　　要：目的:观察高磷诱导的血管平滑肌细胞钙化早期microRNA表达变化,分析其可能参与的信号通路途径。方法:采用高磷(无机磷2.6 mmol/L)刺激大鼠血管平滑肌细胞系A7r5钙化7 d,邻甲酚酞络合酮比色法和考马斯亮蓝法检测细胞内钙含量,RT-PCR法检测平滑肌细胞表型和钙化相关基因的表达变化,茜素红染色观察钙结节。microRNA microarray法检测高磷刺激后0、3、12 h,680种microRNA表达变化。采用TAM软件分析不同时间点间信号激活情况。结果:高磷诱导的A7r5细胞钙盐含量升高9.6倍(P<0.05),平滑肌细胞表型mRNA(SM-αactin,SM22)下调(P<0.05),钙化相关mRNA(BMP2、MSX2、Runx2)上调(P<0.05),茜素红染色后可见高磷刺激组有明显钙结节。680种microRNA在3个时间点的表达各不相同,只有6种microRNA分别逐级上调或渐渐下调。26种信号通路被明显激活,其中包括细胞凋亡、分化增殖等已知与钙化相关的信号通路。结论:microRNA参与调节血管钙化是一种动态微调的过程,为血管钙化研究带来新的思路。Objective： To study the change of microRNA during the early stage of high phosphorus induced vascular smooth muscle cell（ VSMC） calcification and its related mechanism. Methods： The in vitro calcification model was created through stimulating VSMC cell line A7r5 with high Pi（ 2. 6 mmol /L） for 7 d. The calcification was validated through ocresolphthalein complexone colorimetry to detect the cellular calcium content,real-time PCR to measure the calcification-related gene expression and alizarin red staining to observe the formation of calcium nodules. Based on the cell calcification model,microRNA microarray array was applied to screen the profiles of microRNA expression in VSMC following high Pi stimulation for different periods（ 0,3 and 12 h）. The array data were analyzed by TAM tool to explore the activated signaling pathway. Results： The calcium content of A7r5 cells induced by high Pi was increased 9. 6 times high as cells without Pi treatment（ P〈0. 05）. VSMC contractile phenotype genes（ SM-α actin,SM22） were down-regulated（ P〈0. 05）,while calcification-related genes（ BMP2,MSX2,Runx2） were up-regulated（ P〈0. 05） in VSMC stimulated by high Pi. The calcium nodules were obviously formed in cells after 7 d high Pi treatment. In microarray experiment,680 individual microRNAs were detected in high Pi-treated VSMCs at different time points（ 0,3 and 12 h）. Among these genes,miR-183,miR-664 and miR-9＊were increased whereas miR-542-5P,let-7f and miR-29 a were decreased in time-dependent manners. Twenty-six kinds of signaling pathways,including cell apoptosis,differentiation and proliferation,were significantly activated. All these activated pathways were associated with calcification. Conclusion： This study implies that microRNA changed in high Pi-induced VSMCs may involve in the process of calcification.

关 键 词：血管钙化 肌 平滑 血管 微RNA 信号转导 

分 类 号：R331.3[医药卫生—人体生理学]