发卡样CTGF特异性RNA干扰重组体的构建及筛选  

Cloning and screening of the recombinant plasmids affecting gene CTGF by RNA interference

作　　者：主余华[1] 张春清[1] 任万华[1] 马艳丽[1] 赵幼安[2] 

机构地区：[1]山东大学山东省立医院消化内科,山东济南250021 [2]山东大学齐鲁医院消化内科,山东济南250012

出　　处：《山东大学学报（医学版）》2006年第4期351-356,共6页Journal of Shandong University:Health Sciences

基　　金：山东省卫生厅青年基金资助课题(2005年第25号)

摘　　要：目的:利用Pgenesil-1质粒载体构建介导结缔组织生长因子(CTGF)短发夹RNA表达的质粒,并筛选有效的抑制序列。方法:分别设计3对有小发夹结构的两条DNA序列及1对非特异对照序列,经退火成互补双链,再克隆至带有U6启动子的质粒载体Pgenesil-1中,构建重组体,转化DH5α菌株,提取质粒行酶切鉴定后,进行测序分析。将构建成功的4组重组体转染HSC-T6 24 h后,通过半定量RT-PCR分析HSC-T6 CTGFmRNA的表达水平,与空白对照及仅加转染试剂组比较分析。结果:CTGF的shR-NA片段被成功克隆到Pgenesil-1质粒载体中,经酶切与测序证实构建成功,转染HSC-T6 24 h后,与空白对照组相比,通过半定量RT-PCR分析发现有两组细胞CTGFmRNA水平明显下降,24 h抑制效率分别为(74±5)%,(P<0.01);(61±3)%,(P<0.05)。转染非特异shRNA组及仅加转染试剂组CTGFmRNA表达水平无明显变化。结论:成功构建了能表达CTGFshRNA的3组重组体,并筛选出能高效抑制CTGF表达的shRNA序列,为进一步探索肝纤维化基因治疗的新途径奠定了实验基础。Objective：To clone the recombinant plasmids expressing connective tissue growth factor（CT- GF） short hairpin RNA（shRNA） by Pgenesil-1 plasmid, and to screen the highly efficient shRNA. Methods：Three pairs of two DNA sequences containing small hairpin structure and one pair of unspecific control sequence were designed and synthesized respectively. The complement forms were obtained by annealing and inserted into plasmid Pgenesil-1 with U6 promoter, and the recombinant plasmids were transformed into DH5α strain. Finally the plasmids identified by restriction enzyme were used for sequence analysis. Four pairs of recombinant plasmids being transfected into HSC-T6, the expression of CT-GF mRNA level was determined by reverse transcription-polymerase chain reaction 24 hours later. Results： The CrGF shRNAs were successfully inserted into plasmid Pgenesil-1. The recombinants were identified by endonuclease digestion and sequencing. Compared with the controls, the expression of CTGF mRNA level in HSC-T6 transfected with CTGF shRNA recombinants was markedly down-regulated by （74 ± 5 ） % （ P 〈 0.01） and （61 ± 3）%（ P 〈 0.05） respectively in two groups of recombinant plasmids, wherease it had not any significant changes in another shRNA and unspecific shRNA-transfected and only with transfection reagent to HSC-T6. Conclusion： Three pairs of recombinants expreessing CTGF shRNA are successfully constructed, shRNA sequences potently inhibiting CTGF are screened out successfully too, which lends itself to new gene therapy for liver fibrosis.

关 键 词：结缔组织生长因子 RNA干扰 短发夹RNA 质粒 肝纤维化 基因表达 

分 类 号：R575.2[医药卫生—消化系统]