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大鼠microRNA-145慢病毒表达载体的构建及其对血管平滑肌细胞表型转化的影响 被引量:7

Construction of Recombinant Lentivirus Vector Expressing Rno-miR-145 and Its In- fluence on Vascular Smooth Muscle Cells Phenotype Transformation
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摘要 目的构建针对Rno—miR.145的慢病毒表达载体并探讨其在血小板源生长因子(PDGF)诱导的血管平滑肌细胞(VSMC)表型转化中的作用。方法人工合成含有酶切位点粘端埘R-145shDNA双链模板序列,克隆于LV3pGLV/H1/GFP+Puro-miRNA慢病毒穿梭载体中,转染293T细胞,收获并浓缩慢病毒颗粒,感染大鼠原代VSMC,倒置荧光显微镜下观察VSMC感染后的荧光表达情况,实时荧光定量PCR检测miR-145的表达情况;实验分为空白对照组、PDGF组、PDGF+miR-145组和细胞转染阴性慢病毒载体组(miR-NC组);采用实时荧光定量PcR测定miR-145对VsMc增殖相关基因PCNA、C-Jun及分化相关基因SM22amRNA表达水平的影响。结果成功构建了microRNA-145慢病毒载体,测定病毒滴度为1×10^9TU/H1L。倒置荧光显微镜下观察大鼠microRNA.145慢病毒表达载体感染成功,MOI值为50,感染72h时感染率最高。实时荧光定量PCR结果显示PDGF可使PCNA、C—Jun表达增加,而使SM22α表达降低;miR-145可使PDGF诱导的去分化型VSMC增殖相关基因PCNA、C—Jun表达降低,分化相关基因SM22ct表达增加。结论miR-145慢病毒载体可高效感染大鼠原代VSMC。感染miR-145慢病毒后可抑制VSMC的表型转化。 Aim To construct a lentivirus vector expressing microRNA (miRNA) Rno-miR-145 and probe func- tion of the vector in platlet derived growth factor (PDGF) induced vascular smooth muscle cells(VSMC) phenotype trans- form. Methods The miR-145 shDNA double chain template sequence was synthesized artificially and put this tem- plate sequence clone LV3 pGLV/H1/GFP + Puro-miRNA Lentivirus plasmid. 293T cells were transfected. Lentivirus particles(virosome) were harvested and concentrated, then primary cultured VSMC of the rats were infected. Fluores- cence expression infected with VSMC was observed by inverted fluorescence microscope, miR-145 expression condition was detected with real-time PCR. Blank control group,PDGF group, PDGF + miR-145 group and miR-NC group were di- vided in this test. Influence of miR-145 on related genes c-Jun, PCNA, SM22ct expression level was observed with real- time PCR. Results microRNA-145 lentivirus plasmid was construted successfully. The viral titer was 1 × 10^9TU/ mL. microRNA-145 lentivirus expression plasmid was infected successfully. The best transfection efficiency was on the 3th day when multiply infection(MOI) was 50. Real-time PCR results revealed PDGF increased PCNA,c-Jun expression level,but reduced SM22ct expression; miR-145 made VSMC related genes PCNA, c-Jun expression reduce, but made SM22α expression increase. Conclusion MicroRNA-145 lentivirus plasmid may infect rat VSMC efficiently. VSMC phenotype transformation may be inhibited by microRNA-145.
作者 王泽慧 边云飞 卫娜 车星星 肖传实 WANG Ze-Hui, BIAN Yun-Fei,WEI Na, CHE Xing-Xing, and XIAO Chuan-Shi ( Shanxi Medical University, Taiyuan, Shanxi 030001, China)
出处 《中国动脉硬化杂志》 CAS CSCD 北大核心 2012年第5期 424-428,共5页 Chinese Journal of Arteriosclerosis
关键词 microRNA-145 慢病毒表达载体 血管平滑肌细胞 表型转化 MicmRNA-145 Lentivirus Expression Plasmid Vascular Smooth Muscle Cells(VSMC) Pheno-type Transformation
作者简介 [作者简介]王泽慧,硕士研究生,研究方向为冠心病的基础与临床,E—mailwangzehui085@sina.com。 边云飞,博士,硕士研究生导师,主要研究方向为冠心病基础与临床,E-mailyunfeibian@sina.com。 通讯作者肖传实,博士,博士研究生导师,研究方向为冠心病基础与临床,E-mailganxibaozhongxin@sina.com。
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