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miR-145 shDNA双链模板序列,克隆于LV3 pGLV/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SM22αmRNA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1×109TU/mL。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72 h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SM22α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.
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
