摘要
【目的】研究micro RNA-1(miR-1)参与切应力条件下ECs(endothelial cells,ECs)对VSMCs(vascular smooth muscle cells,VSMCs)增殖活性调控的力学生物学反应机理,将有利于进一步探索动脉粥样硬化等心血管疾病的发病机制。【方法】应用细胞联合培养平行平板流动腔系统对与VSMCs联合培养的ECs施加15 dyn/cm2的正常切应力(normal shear stress,NSS)和5 dyn/cm2的低切应力(low shear stress,Low SS),加载时间为12 h;Western blotting检测联合培养VSMCs的细胞核增殖抗原(proliferating cell nuclear antigen,PCNA)表达变化;实时荧光定量PCR检测联合培养VSMCs的miR-1表达变化;通过Target Scan、miRWalk等网站预测miR-1的目标蛋白;Western blotting检测联合培养VSMCs的miR-1靶蛋白锌指转录因子4(kruppel-like factor 4,Klf4)的表达;通过转染技术,上调和抑制VSMCs的miR-1表达,Western blotting技术检测靶蛋白Klf4和PCNA的表达变化,以此验证miR-1对Klf4的负调控作用。【结果】与NSS相比,Low SS促进联合培养VSMCs的PCNA表达并显著抑制miR-1表达;Target Scan、miRWalk等网站预测miR-1的下游靶蛋白为Klf4;与NSS相比,Low SS明显上调联合培养VSMCs的miR-1靶蛋白Klf4的表达;静态条件下上调VSMCs的miR-1表达,靶蛋白Klf4和PCNA表达均显著降低,抑制VSMCs的miR-1表达,靶蛋白Klf4和PCNA表达均明显升高。【结论】Low SS通过联合培养VSMCs的miR-1及靶蛋白Klf4,促进VSMCs的增殖。
【Objective】To research the role of micro RNA-1（miR-1）on the proliferation of vascular smooth muscle cells（VSMCs）induced by endothelial cells（ECs）under low shear stress（Low SS）,which will help to find the pathogenesis of atherosclerosis and some other cardiovascular diseases.【Methods】By using co-culture parallel plate flow chamber system, ECs and VSMCs were co-cultured and applied to normal shear stress（NSS）（15 dyn/cm2） and low shear stress（Low SS）（5 dyn/cm2） for 12 h. Real-time PCR was used to examine the levels of miR-1 in the co-cultured VSMCs. The target protein of miR-1 was predicted by Target Scan, miRWalk and some other websites. The expressions of kruppel-like factor 4（Klf4） and proliferating cell nuclear antigen（PCNA） in the co-cultured VSMCs were detected by Western blotting. To verify the negative regulation of miR-1 on Klf4, mimics and inhibitors were used to alter the expression of miR-1, and then the expression of Klf4 and PCNA was detected by Western blotting.【Results】Compared with NSS,Low SS increased the PCNA expression in the co-cultured VSMCs while significantly inhibited the miR-1 expression. It was predicted in Target Scan, miRWalk and other websites that miR-1 could regulate the expression of Klf4. Compared with NSS, Low SS could significantly upregulate the Klf4 expression in the co-cultured VSMCs. In the static, the expression of miR-1 was downregulated in VSMCs, which resulted in a significant increase of the Klf4 and PCNA expression, and miR-1 overexpression led to a decrease of the Klf4 and PCNA expression.【Conclusion】Low SS can promote the proliferation in co-cultured VSMCs via miR-1 and its target protein Klf4.
作者
王聪聪
徐忠伟
李霞
WANG Cong-cong;XU Zhong-wei;LI Xia(Department of Health Services,Logistics University of PAP,Tianjin 300309,China)
出处
《武警后勤学院学报（医学版）》
CAS
2018年第3期190-194,共5页
Journal of Logistics University of PAP（Medical Sciences）
基金
武警后勤学院博士启动基金(WHB201506)
