目的構(gòu)建針對結(jié)締組織生長因子（connective tissue growth factor,CTGF）的RNA干擾重組質(zhì)粒及其對照質(zhì)粒,將其轉(zhuǎn)染到體外培養(yǎng)的大鼠肝星狀細胞（Hepatic Stellate Cells,HSC）中,進一步應(yīng)用Real-time PCR方法檢測CTGFmRNA的表達,觀察RNA干擾對大鼠肝星狀細胞CTGF表達的抑制作用,為臨床基因治療提供依據(jù)。方法①利用pGCsi載體構(gòu)建介導(dǎo)CTGF短發(fā)夾RNA（shRNA）表達的質(zhì)粒重組體,并通過基因測序分析進行鑒定。②將構(gòu)建成功的4組重組質(zhì)粒轉(zhuǎn)染大鼠肝星狀細胞株（HSC-T6）,另設(shè)空白對照組,48 h后,共聚焦顯微鏡檢測綠色熒光蛋白（green fluorescent protein,GFP）表達,分析轉(zhuǎn)染效果。③檢測CTGFmRNA表達水平,篩選出有效抑制CTGF表達的序列。④應(yīng)用SPSS 17.0軟件對數(shù)據(jù)進行方差分析。結(jié)果成功構(gòu)建了能表達CTGFshRNA的三組重組體pGCsi-CTGFshRNA。pGCsi-CTGFshRNA成功轉(zhuǎn)染到肝星狀細胞中。與空白對照組相比,pGCsi-CTGFshRNA1組及pG... 

【文章來源】：中國醫(yī)學(xué)工程. 2013,21(05)

【文章頁數(shù)】：4 頁

【部分圖文】：

質(zhì)粒構(gòu)建載體示意圖

.Connectivetissuegrowthfactormediatestransforminggrowthfactorbeta-inducedcollagensynthesis:down-regulationbycAMP[J].FASEBJ,1999,13(13):1774-1786.[5]SedlaczekN,JiaJD,BauerM,etal.Proliferatingbileductepithelialcellsareamajorsourceofconnectivetissuegrowthfactorinratbiliaryfibrosis[J].AmJPathol,2001,158(4):1239-1244.[6]GeorgeJ,TsutsumiM.siRNA-mediatedknockdownofconnectivetissuegrowthfactorpreventsN-nitrosodimethylamine-inducedhepaticfibrosisinrats[J].GeneTher,2007,14(10):790-803.圖2pGCsi-CTGFshRNA1質(zhì)粒(a)，pGCsi-CTGFshRNA2質(zhì)粒(b)，pGCsi-CTGF-shRNA3質(zhì)粒(c)及pGCsi-CTGFshRNAHK質(zhì)粒(d)(陰性對照質(zhì)粒)分別轉(zhuǎn)染HSC-T648h后綠色熒光蛋白表達檢測結(jié)果。abcd

.Connectivetissuegrowthfactormediatestransforminggrowthfactorbeta-inducedcollagensynthesis:down-regulationbycAMP[J].FASEBJ,1999,13(13):1774-1786.[5]SedlaczekN,JiaJD,BauerM,etal.Proliferatingbileductepithelialcellsareamajorsourceofconnectivetissuegrowthfactorinratbiliaryfibrosis[J].AmJPathol,2001,158(4):1239-1244.[6]GeorgeJ,TsutsumiM.siRNA-mediatedknockdownofconnectivetissuegrowthfactorpreventsN-nitrosodimethylamine-inducedhepaticfibrosisinrats[J].GeneTher,2007,14(10):790-803.圖2pGCsi-CTGFshRNA1質(zhì)粒(a)，pGCsi-CTGFshRNA2質(zhì)粒(b)，pGCsi-CTGF-shRNA3質(zhì)粒(c)及pGCsi-CTGFshRNAHK質(zhì)粒(d)(陰性對照質(zhì)粒)分別轉(zhuǎn)染HSC-T648h后綠色熒光蛋白表達檢測結(jié)果。abcd

【參考文獻】：
期刊論文
[1]抗結(jié)締組織生長因子小分子干擾RNA防治大鼠肝纖維化研究[J]. 李光明,謝青,史毅,李定國,張明均,徐芹芳,姜山,周惠娟,金由辛.  中華消化雜志. 2005(06)



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