文章摘要
刘苏健,刘 宏,赵 京,申庆民,常 程,邓勇志,马 捷.大鼠Pik3cb短发卡状RNA对平滑肌细胞增殖的影响[J].南京医科大学学报,2008,28(10):1234~1239
大鼠Pik3cb短发卡状RNA对平滑肌细胞增殖的影响
The construction of rat Pik3cb shRNA and its inhibitory effect on the proliferation of smooth muscle cell
投稿时间:2008-05-20  
DOI:10.7655
中文关键词: 载体构建  Pik3cb  shRNA  血管再狭窄  RNAi
英文关键词: vector construction/Plasmid  Pik3cb  shRNA  restenosis  RNAi
基金项目:
作者单位
刘苏健 北京航天总医院胸外科,北京 100076 
刘 宏 北京航天总医院胸外科,北京 100076 
赵 京 北京航天总医院胸外科,北京 100076 
申庆民 北京航天总医院胸外科,北京 100076 
常 程 北京航天总医院胸外科,北京 100076 
邓勇志 山西医科大学第二医院心胸外科,山西 太原 030001 
马 捷 山西医科大学第二医院心胸外科,山西 太原 030001 
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中文摘要:
      目的:构建大鼠Pik3cb短发卡状RNA(shRNA)真核表达载体,为利用RNA干扰(RNA interference,RNAi)技术从转录后水平抑制血管移植术后移植静脉再狭窄的研究做准备?方法:经预实验设计并合成6条shRNA寡核苷酸片段,酶切鉴定和测序正确后挑选2条质粒转染入大鼠胸主动脉平滑肌细胞内,48?72 h用荧光显微镜观察转染效率,Western blot检测Phospho-Akt(Ser473)蛋白的表达,流式细胞仪分别在24?48?72?96 h检测细胞凋亡数目?结果:构建的两条大鼠Pik3cb shRNA经限制性内切酶酶切和DNA测序正确后分别命名为pU6-Pik3cb-shRNA-1和pU6-Pik3cb-shRNA-2,转染平滑肌细胞48 h和72 h转染率分别为15.7%和10.1%;Western blot结果显示转染组Phospho-Akt(Ser473)蛋白表达明显下降;流式结果表明pU6-Pik3cb-shRNA-1和pU6-Pik3cb-shRNA-2组凋亡细胞数目明显增加,与对照组相比有统计学意义(P < 0.05),各时间点错配质粒组与正常对照组相比差异无统计学意义?结论:成功构建了2条大鼠Pik3cb shRNA真核表达载体,有效促进凋亡,抑制平滑肌细胞增殖,为靶向Pik3cb的RNAi防治血管再狭窄奠定了基础?
英文摘要:
      Objective:To construct rat Pik3cb(phosphatidylinositol 3-kinase,catalytic,beta polypeptide) shRNA eukaryon plasmid express vector,and test its downregulating effect on Pik3cb mRNA expression for the application of RNA interference in restraining vein graft restenosis. Methods:Two shRNA of rat Pik3cb were design and synthesize according to the sequence of Pik3cb in the Genbank,annealed to form double strands and then cloned into pGenesil-1,and then the sequences were examed. After shRNAs were transfected into rat thoracic VSMC through METAFECTENETM,the transfection rate was identified by EGFP. pAkt-473 protein expression was detected by Western blot,and cell apoptosis were obtained through flow-cytometry. Results:The two sequences of synthesized shRNA named pU6-Pik3cb-shRNA-1 and pU6-Pik3cb-shRNA-2 were exactly the same with the design. The 48 h and 72 h transfection rate were 15.7% and 10.1%,respectively. The pAkt-473 protein expression significantly reduced,while the apoptosis cells were more significantly increased in the shRNA groups than the control groups(P < 0.05). The scramble shRNA did not interfere with the VSMC proliferation. Conclusion:Rat Pik3cb shRNA eukaryon express plasmid vectors were constructed successfully and effectively inhibited the proliferation of rat smooth mouse cells.
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