摘要
目的探讨纤维介素蛋白(fg12)的双链RNA(dsRNA)在体外对fg12凝血酶原酶基因表达的干扰作用及其规律。方法构建能产生鼠垃12(mfg12)的发夹状双链RNA(shRNA)的载体p-mfg12shRNA,将其与mfg12- EGFP融合基因表达质粒pEGFP-mfg12共转染(用量比为1:5)到中国仓鼠卵巢细胞(CHO细胞)作为实验组,另仅转染pEGFP-mfg12或共转染无关序列shRNA表达质粒和pEGFP-mfg12(用量比为1:5)作为对照组。转染24、48、 72 h后,观察mfg12-EGFP融合蛋白在CHO细胞中的表达情况,并通过流式细胞仪检测荧光细胞阳性率。分别将p- mfg12shRNA和mfg12cDNA表达质粒pcDNA3．1-mfg12共转染(用量比为1:5)到CHO细胞和人宫颈癌细胞(Hela 细胞),作为试验组,另转染pcDNA3．1-mfg12或共转染无关序列shRNA表达质粒和pcDNA3．1-mfg12(用量比为1:5) 或不转染任何质粒作为对照组。通过逆转录聚合酶链反应和免疫组织化学检测mfg12在CHO和Hela这两种细胞株中表达的情况。结果实验组较对照组的绿色荧光强度明显减弱,荧光细胞数明显减少。在CHO和Hela两种细胞株,均显示mfg12shRNA显著抑制mfg12 mRNA和蛋白质的表达。结论本研究所构建的mfg12shRNA表达质粒p-mfg12shRNA 可在体外高效特异地抑制mfg12的表达,为进一步的体内实验奠定了基础。
Objective To construct the sLRNA plasmid for mfg12 gene, which has been reported to be involved in a variety of disease developments including fulminant viral hepatitis, acute rejection of allo/zero transplantation and fetal loss syndrome, and to investigate its inhibitory effects on mfg12 expression in vitro. Methods A plasmid p-mfg12shRNA complimentary to the sequerence responsible for the functional domain of mouse fg12 （mfg12） was constructed. The pcDNA3.1 mfg12 expression construct was able to show a satisfactory fg12 protein expression. The plasmid expression pEGFP and a construct expressing irrelevant shRNA with a random combination of the p-mfg12shRNA sequence were used as controls. A pEGFP-mfg12 expressing mfg12-EGFP fusion protein was also constructed for screening of the effect of p-mfg12shRNA on the mfg12 expression. Results Cotransfection of p-mfg12shRNA with pEGFP-mfg12 decreased green fluorescent cells and the tightness of fluorescence within the cells at the 24 h, 48 h and 72 h post-transfection when compared with that in the control groups which were solely lransfected with pEGFP-mfg12. Furthermore the mfg12 expression was significantly reduced when the pcDNA3.1 mfg12 expression construct was cotransfected with p-mfg12shRNA both at mRNA level by RT-PCR and protein level by RT-PCR, immtmohistochemistry staining and FACS in both CliO cell and Hela cell lines. Condnsion The study demonstrated that the construct of p-mfg12shRNA successfully interfered in the mfg12 expression in vitro. It provides a basis for a further investigation of effect in vivo.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2006年第5期358-363,共6页
Chinese Journal of Hepatology
基金
国家杰出青年科学基金(30225040)
武汉市科技攻关项目(20027006144)
