腺病毒载体RNAi技术抑制血吸虫病小鼠肝细胞Fas表达

Recombinant adenovirus vector with RNAi technique inhibits the Fas expression in hepatic cells of mice with schistosomiasis

目的探讨RNAi技术对血吸虫病肝组织中Fas表达的作用。方法采用尾静脉注射腺病毒载体,在小鼠体内表达Fas-shRNA抑制血吸虫病小鼠肝细胞Fas的表达。冰冻切片荧光显微镜检查腺病毒载体对肝细胞的转染率,免疫组化和Western Blot方法检测肝组织Fas表达情况,RT-PCR方法检测肝组织Fas mRNA表达情况。结果免疫组化和Western Blot方法检测均表明RNAi组Fas表达受到抑制(t=29.799,P<0.01),模型组和HK对照组Fas表达增强。对Fas mRNA表达的RT-PCR方法检测也与上述结果一致。腺病毒载体对肝细胞的转染率达78.5%。结论腺病毒载体Fas-shRNA能够有效抑制血吸虫病小鼠肝细胞Fas的表达,尾静脉注射腺病毒载体能高效率感染肝细胞。

To investigate the inhibitory effect of the recombinant adenovirus vector with Fas（CD95）-targeted shRNA on the Fas-expresstion in hepatic cells of mice, with schistosomiasis, BALB/c mice were injected via the tail vein with the recombinant adenovirus vector pAdeno-X-siFasl＋siFas2 expressing two Fas-targeted short hairpin RNAs （shRNA）. The staining of Fas was performed by immunohistochenical methods in routine paraffin-embeded hepatic sections and the transfection rate of hepatic cells with the recombinant adenovirus vector was determined by the fluorescence microscopy examination of the freezing section of livers. Western blot assay was carried out for appraising the expression of Fas protein, and RT-PCR was undertaken for the detection of Fas mRNA. It was demonstrated either by immunohistochemical stainings or Western blot assay that the expression of Fas in group of mice treated with RNAi technique were inhibited （t= 29. 799, P〈0. 01）, while those in model group and HK control group were enhanced in comparison with that of mice treated with RNAi technique. In addition,the results from RT-PCR assay were consistent with those of immunohistchemistry and Western blot assay. The transfection rate of the recombinant adenovirus vector to hepatic cells was up to 78. 5%. It is apparent that recombinant adenovirus vector FasshRNA can inhibit effectively the Fas expression in hepatic cells of mice with schistsomiasis.