小鼠Fas及TNFR1的miRNA表达质粒的构建及体内外干预的初步研究  

Construction and Inhibition Effect in vitro and in vivo of MiRNA against Mouse Fas and TNFR1

作　　者：王鸣[1] 叶华丽[1] 刘君慧[1] 陈佳佳 习东[1] 宁琴[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院感染性疾病研究所,武汉430030

出　　处：《中国药师》2018年第9期1513-1517,共5页China Pharmacist

基　　金：教育部创新团队发展计划滚动支持项目(编号:IRT14R20)

摘　　要：目的:探讨针对小鼠Fas及TNFR1的miRNA表达质粒在体外对相应基因表达的干预效应及体内分别对重型肝炎小鼠的治疗作用。方法:分别构建产生小鼠Fas-miRNA、TNFR1-miRNA的表达载体P-m Fas-miRNA、P-m TNFR1-miRNA,将其与相应表达质粒(pc DNA3.1-m Fas或pc DNA3.1-m TNFR1)分别共转染到中国仓鼠卵巢细胞(CHO细胞)中。通过实时荧光定量PCR及Western Blot观察Fas或TNFR1在转录水平及蛋白水平表达量的改变。建立重型肝炎小鼠模型,通过尾静脉高压注射P-m FasmiRNA或P-m TNFR1-miRNA,分别观察P-m Fas-miRNA或P-m TNFR1-miRNA对重型肝炎小鼠的治疗作用。结果:成功构建了针对小鼠Fas及TNFR1的miRNA。在CHO细胞中,干预组Fas及TNFR1在RNA水平及蛋白水平的表达均较对照组显著降低。Pm Fas-miRNA、P-m TNFR1-miRNA分别提高重型肝炎小鼠生存率至11.1%和20%。结论:Fas及TNFR1可作为重型肝炎基因治疗的靶点,利用miRNA沉默Fas及TNFR1基因将有利于重型肝炎的治疗,为后续的进一步研究奠定了基础。Objective： To construct miRNA targeted mouse Fas （mFas） and mouse TNFR1 （mTNFRI） and explore their inhibi- tion effect in vitro and in the treatment of fulminant hepatitis mouse model. Methods： MiRNA expression plasmid against mFas or mT- NFR1 （P-mFas-miRNA or P-mTNFRI-miRNA） was constructed, and then cotransfected with pcDNA3.1-mFas expression plasmid or pcDNA3.1- mTNFR1 expression plasmid into CHO cells. The levels of mFas or mTNFR1 mRNA transcript and protein were analyzed by RT-PCR and Western Blot. Meanwhile, the fulminant hepatitis model induced by murine hepatitis virus strain 3 （ MHV-3 ） was es- tablished and P-mFas-miRNA or P-mTNFRI-miRNA were transfeeted into mouse hepatocytes by hydrodynamic tail vein injection, and then the survival rate of fulminant hepatitis mice was observed. Results： P-mFas-miRNA and P-mTNFRI-miRNA were constructed successfully, which could downregnlate mFas or mTNFR1 gene and protein expression significantly. Furthermore, P-mFas-miRNA and P-mTNFRI-miRNA both protected mice from fulminant hepatitis when compared with the control group. Conclusion： Fas and TNFR1 might be targets for gene therapy in fulminant hepatitis. Fas and TNFR1 silencing using miRNA may be useful in treating fulminant hepatitis.

关 键 词：重型肝炎 RNA干扰 FAS TNFR1 microRNA 

分 类 号：R968[医药卫生—药理学]