摘要
目的探索人fg12凝血酶原酶（hfg12）短干扰RNA（siRNA）在体外对hfg12凝血酶原酶基因表达的干预效应。方法构建产生hfg12短发夹状RNA（shRNA）的载体Phfg12shRNA，将其与hfg12-增强型绿色荧光蛋白（EGFP）融合基因表达质粒pEGFPhfg12共转染到中国仓鼠卵母细胞（CHO细胞），转染48h后，倒置荧光显微镜下观察EGFP在CHO细胞中的表达，并通过流式细胞仪检测荧光细胞阳性率。将Phfg12shRNA和hfg12表达质粒pcDNA3．1-hfg12共转染到CHO细胞，通过实时荧光定量PCR和免疫组织化学检测hfg12基因转录和蛋白表达水平的变化。分为4组：共转染P—hfg12shRNA和pcDNA3．1-hfg12为干预组，共转染无关序列shRNA表达质粒和pcDNA3．1-hfg12为非相关干预组，仅转染pcDNA3．1-hfg12为未干预组，不转染任何质粒为空白组。结果在CHO细胞中，含Phfg12shRNA的干预组较未干预组和非相关干预组的绿色荧光强度明显减弱，荧光细胞数明显减少；荧光表达阳性细胞率：干预组α为6．5％±0．2％，未干预组α为40．1％±1．8％，两组比较，Χ^2=8．056，P〈0．01，差异有统计学意义。抑制效率达85．5％。hfg12shRNA显著抑制了hfg12mRNA和蛋白质的表达，干预组hfg12mRNA水平为0．11±0．01，未干预组为0．84±0．06，两组比较，F=10．7，P〈0．01，差异有统计学意义。结论P—hfg12shRNA可在体外高效特异地抑制hfg12基因转录和蛋白的表达，为进一步的体内干扰实验奠定了基础。
Objective Our previous studies have shown that an anti-sense plasmid to mouse fibrinogen like protein 2 （mfg12） significantly reduced mfg12 expression in vivo, markedly ameliorated inflammatory infiltration, fibrin deposition and hepatocyte necrosis, prolonged the survival time period and elevated the survival rate in Balb/cJ mice with murine hepatitis virus type 3 （MHV-3） induced fulminant hepatitis. This study was designed to explore the opportunity of RNA interference technique in the inhibitory application of hfg12 expression, which has been reported to be involved in a variety of disease developments including fulminant viral hepatitis, acute rejection of allo/xeno transplantation and fetal loss syndrome. Methods A plasmid named p-hfg12shRNA complimentary to the sequence responsible for hfg12 was constructed; meanwhile irrelevant shRNA plasmid with a random combination of the p-hfg12shRNA sequence was used as a control. A plasmid named pEGFP-hfg12 expressing hfg12-EGFP fusion protein was also constructed for the screening of the effect ofp-hfg12shRNA on the hfg12 expression. By cotransfection of the constructed p-hfg12shRNA and pEGFP-hfg12 or pcDNA3.1-hfg12 expression plasmids into CHO cells, the inhibition of hfg12 expression by hfg12shRNA was analyzed by direct observation through fluorescent microscopy, FACS, real time PCR and immunohistochemistry staining. Results The experiments showed a significant inhibitory effect ofp-hfg12shRNA on hfg12 expression at 48 h post-cotransfection by observation of green fluorescent cells, FACS, real time PCR and immunohistochemistry staining, with the inhibitory efficiency reaching as high as 85.5%. Conclusion The study demonstrated that p-hfg12shRNA successfully interfered with hfg12 expression in vitro. This provides a foundation to further explore its application in vivo.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2008年第12期930-934,共5页
Chinese Journal of Hepatology
基金
国家自然科学基金（30571643、30672380、30700702）
国家重点基础研究发展计划（“973”）项目
（2007CB512900）
“十一五”国家科技支撑计划项目（2006BAI05A07）
卫生部部属管医院临床学科重点项目
