小鼠纤维介素蛋白2微小RNA表达质粒的构建及其对体内外相应基因的干预效应

Construction and inhibition effect of miRNA against mouse fgl2

目的:探讨小鼠纤维介素蛋白2(fgl2)的miRNA(微小RNA)表达质粒在体外对相应基因表达的干预效应及在体内对重型肝炎小鼠的治疗作用。方法:分别构建产生小鼠fgl2-miRNA的表达载体(P-mfgl2-miRNA),将其转染到中国仓鼠卵巢(CHO)细胞中。实验分组:将P-mfgl2-miRNA与小鼠fgl2的表达质粒(pcDNA3.1-mfgl2)共转染到CHO细胞作为干预组,无关序列miRNA表达质粒(irrelevant P-miRNA)与pcDNA3.1-mfgl2共转染为非相关对照组,仅转染pcDNA3.1-mfgl2为阳性对照组,未转染任何质粒为空白对照组。通过实时荧光定量PCR及Western blot观察fgl2在基因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-mfgl2-miRNA者为治疗组,注射irrelevant P-miRNA者为非相关组,注射相同剂量生理盐水者为阴性对照组,观察P-mfgl2-miRNA对重型肝炎小鼠的治疗作用。结果:成功构建了针对小鼠fgl2的miRNA。在CHO细胞中,干预组fgl2在RNA水平及蛋白水平的表达均较非相关对照组显著降低。P-mfgl2-miRNA可提高重型肝炎小鼠生存率至8.3%。结论:Fgl2可作为重型肝炎基因治疗的靶点,利用miRNA沉默fgl2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 fgl2 （mfgl2） and explore its inhibition effect in vitro and effect on the treatment of fulminant hepatitis mice model. Methods ： MiRNA expression plasmid against mfgl2 （P-mfgl2-miRNA） was constructed, then it was transfected into CHO cells. Experimental group： @Interference group： P-mfgl2-miRNA and pcD- NA3. 1- mfgl2 expression plasmid were cotransfected into CHO cells. ~）Irrelevant control group： irrelevant sequence miRNA ex- pression plasmid and pcDNA3. 1-mfgl2 expression plasmid were cotransfected into CHO cells. （~）Positive control group： only pcDNA3.1- mfgl2 expression plasmid was transfected. @Blank control group. The level of mfgl2 mRNA transcript and protein were analysed by RT-PCR and Western blot. Besides, the fulminant hepatitis model induced by murine hepatitis virus strain 3 （MHV-3） was established and the mice were randomly assigned to three group： P-mfgl2-miRNA as treatment group, irrelevant sequence miRNA plasmid as irrelevant control group and blank control group. Plasmids refered above was transfected into mouse hepatocytes in vivo by hydrodynamic tail vein injection and then survival rate of fulminant hepatitis mice was observed. Results： P-mfg]2-miRNA was constructed successfully, which could down-regulate mfgl2 gene and protein expression significantly, P-mf- g12- miRNA can protect mice from fulminant hepatitis, as compared with control groups. Conclusion： Fgl2 might be target for gene therapy in fulminant hepatitis. Fgl2 silencing using miRNA may be useful for treating fulminant hepatitis.