【作者】 彭韦霞；

【导师】 文芳；

【作者基本信息】 南华大学 ， 内分泌内科， 2007， 硕士

【摘要】 目的利用RNA干扰技术,研究小凹蛋白（Caveolin-1）对胰岛素刺激的人脐静脉内皮细胞（ECV304细胞）纤维蛋白溶解酶原抑制物-1（Plasminogen Activator Inhibitor-1,PAI-1）表达的影响。方法通过Ambion专用软件设计合成两条编码shRNA的互补DNA寡核苷酸单链,退火形成双链核苷酸。通过T4连接酶将双链克隆入含RNA PolIII聚合酶表达元件的pENTR^TM/U6入门载体后测序,利用Gateway技术构建Caveolin-1 RNAi慢病毒表达载体。运用实时定量RT-PCR、间接免疫荧光、Western-blot分析ECV304细胞Caveolin-1基因沉默效率。然后用生理浓度胰岛素(终浓度1×10^-9mol/L)和高浓度胰岛素(终浓度1×10^-7mol/L)处理ECV304细胞,检测Caveolin-1基因沉默前后不同浓度胰岛素处理的ECV304细胞中PAI-1mRNA和蛋白表达和不同浓度胰岛素处理后Caveolin-1mRNA和蛋白表达变化。结果1.测序结果显示插入pENTR^TM/U6载体中的编码Caveolin-1基因shRNA序列的U6RNAi盒大小及阅读框架正确,Caveolin-1 pENTR^TM/U6入门载体构建成功。U6上游引物与V5下游引物PCR鉴定Caveolin-1 RNAi慢病毒表达载体,琼脂糖凝胶电泳片段与目的片段大小一致。2、实时定量RT-PCR、间接免疫荧光、Western-blot分析入门载体、RNAi表达载体对Caveolin-1基因抑制效率达85%,且特异性较高,对无关基因如β-actin无影响,同时沉默无关基因S100A13不能干扰Caveolin-1表达。3、高胰岛素可促使PAI-1mRNA和蛋白表达明显增加,在高胰岛素刺激PAI-1表达的同时,细胞中的Caveolin-1表达明显降低,在RNA干扰Caveolin-1基因后高胰岛素促PAI-1表达的作用更加明显;生理浓度胰岛素处理ECV304细胞后,PAI-1的表达增加,Caveolin-1表达无变化,shRNA诱导Caveolin-1沉默后生理浓度胰岛素并不明显增加PAI-1的表达。结论1、成功构建了针对Caveolin-1基因的RNAi入门载体和表达载体,能有效而又特异地抑制Caveolin-1基因表达达85%。2、Caveolin-1可能并不参与生理状态胰岛素促PAI-1表达的信号转导,高胰岛素则可能通过抑制Caveolin-1基因的表达来促使PAI-1的表达更多还原

【Abstract】 【Objective】The aim of this study is to investigate how Caveolin-1 gene affects the expression of insulin-stimulated Plasminogen Activator Inhibitor-1 （PAI-1） in human vascular endothelial cells （ECV304） by RNA interfering technology.【Method】Two complementary oligonucleotide strand coding small hair RNA for Caveolin-1 was designed by using siRNA Target Finder from Ambion. The Caveolin-1 shRNA double strand oligonucleotide was cloned into the pENTR^TM/U6 entry vector containing RNA Polymerase III express element by T4 ligase and then the Caveolin-1 RNAi lentiviral expression system was successfully constructed by utilizing gateway technology. To evaluate the efficiency of Caveolin-1 RNAi rate, Real-time RT-PCR, immunofluorescence staining and Western blot were used to analyze the Caveolin-1 mRNA and protein expression after ECV304 cells were transfected with Caveolin-1-shRNA. By utilizing Caveolin-1 RNAi expression system, we were able to compare the mRNA and protein levels of PAI-1 between ECV304 cells and Caveolin-1RNAi ECV304 cells after they cultured with physiological concentration of insulin (final concentration 1×10^-9)and high concentration(final concentration 1×10^-7) as well as the mRNA and protein levels of Caveolin-1 stimulated by different concentration of insulin.【Results】1.The size and sequence of Caveolin-1 U6 RNAi cassette insert in the pENTR?/U6 vector was confirmed by sequencing analysis.PCR were used to identifying the BLOCK-iT^TM Lentiviral RNAi Expression vector by U6 sense primer and V5 antisense. The PCR product analysed by electrophoresis was correct.2.The mRNA and protein expression of Caveolin-1 decreased 85% measured by REAL-TIME RT-PCR, immunofluorescence staining and Western blot. Other genes, such as S100A13 andβ-actin did not affect the interfering course.3.High insulin increase the mRNA and protein expression of PAI-1 whereas high insulin treatment suppressed Caveolin-1 gene expression in ECV304 cells. In contrast, PAI-1 mRNA and protein level was augmented dramatically in the Caveolin-1 RNAi ECV304 cells cultured in the high insulin;while cells were cultured in the physiology insulin culture,Caveolin-1 gene expression had no change, but PAI-1 did not increase when Caveolin-1 RNAi ECV304 cells were treated with physiology dose of insulin.【Conclusions】1. The Caveolin-1 RNAi pENTR^TM/U6 vector and Lentiviral RNAi Expression vector were constructed successfully.The shRNA can inhabit the expression of Caveolin-1 gene efficiently and specially ,the inhabit rate reach 85%.2. Perhaps,PAI-1 expression is a Caveolin-1 dependent in the pathophyiological conditions such as hyperinsulinism but not in the physiological ones.更多还原