乙型肝炎病毒特异性小干扰RNA对转基因小鼠毒副作用的评价

Evaluation of Specific Hepatitis B Virus Small Interfering RNA on Adverse Effect of Transgene Mice

目的探讨基于载体的表达性小干扰RNA(siRNA)对乙型肝炎病毒(HBV)转基因小鼠的毒副作用。方法 siRNA表达载体经微静脉注射转染HBV转基因小鼠,设立特异性siRNA组、PBS对照组、阴性质粒对照组及正常Balb/c小鼠对照组。在注射后5 d、12 d、19 d、26 d、33 d、2个月和3个月的不同时间,经其内眦静脉采血检测小鼠ALT水平变化。ELISA方法检测其血清干扰素-α(IFN-α)、肿瘤坏死因子-α(TNF-α)水平。W estern blot检测其肝脏组织中B细胞淋巴瘤2(Bcl-2)、原癌基因(c-Fos)、Bcl-2相关X蛋白(Bax)的表达变化。采用TUNEL染色检测细胞凋亡,HE染色进行组织病理学观察。结果基于聚合酶Ⅲ(PolⅢ)的siRNA表达载体与基于聚合酶Ⅱ(PolⅡ)的siRNA表达载体相比,长期作用机体可使转基因小鼠血清ALT水平显著升高(P<0.05);ELISA分析发现IFN-α水平前者似乎高于后者,但差异无统计学意义(P>0.05)。在治疗26 d,直到治疗3个月,特异性siRNA治疗组与PBS对照组相比,TNF-α表达在PolⅢ组表达明显降低(P<0.05);而PolⅡ组TNF-α表达与PBS对照组比较差异无统计学意义。W estern blot结果表明肝组织Bcl-2、c-Fos表达显著增加,而Bax表达明显降低(P<0.05),且该组凋亡分析表明细胞凋亡被明显抑制。组织形态学可见肝细胞出现二倍体,核仁增大,核膜清晰可见。结论基于PolⅢ的siRNA表达载体与PolⅡ的siRNA表达载体相比对肝细胞有一定的毒副作用,凋亡被抑制,细胞增殖失控。过强表达的HBV特异性siRNA可影响机体的生物网络调控,导致增殖与凋亡的失衡。

Objective To explore the adverse effects of vector based small interfering RNA（siRNA） on the hepatitis B virus（HBV） transgene mice.Methods The constructed siRNA expressed vectors was transfected HBV transgene mice by hydrodynamics-based injection via vena caudalis.The normal Balb/c mice and transgene mice injected with phosphate buffered solution and negative plasmid were set as controls.The effect was observed in different periods（5 d,12 d,19 d,26 d,33 d,2 months and 3 months after injection）.The plasma alanine transarninase（ALT） level was detected.Interferon-α（IFN-α） and tumor necrosis factor-α（TNF-α） were detected by enzyme linked immunosorbent assay.The Western blot was performed to detect the changes in Bcl-2,c-Fos and Bax in the liver tissue.The changes in apoptosis and histopathology of liver cells were demonstrated by TUNEL and HE staining.Results Compared with the Pol Ⅱ expression vectors,the Pol Ⅲ expression vectors could raise the plasma ALT level（P0.05）.There were no significant differences in IFN-α between the pSilencer 3.1/siRNA group and pSilencer 4.1/siRNA group（P0.05）.But the TNF-α level obviously decreased after treatment with pSilencer 3.1 for 26 d compared with the controls.While there was no difference between the Pol Ⅱ groups and the controls till 3 months.Moreover,the expression of Bcl-2 and c-Fos markedly increased,while the Bax was significantly declined in the pSilencer 3.1/siRNA group（Pa0.05）.The changes resulted in the abnormal proliferation and the depressant apoptosis of hepatocytes.The histomorphology also demonstrated that the hepatocytes had appeared diploid,big nucleoli and distinct nuclear membrane.Conclusions Compared with the Pol Ⅱ driven siRNA,the Pol Ⅲ driven siRNA has toxicity effect on the hepatocytes,the apoptosis is inhabited and the proliferation is out of control.It seems that the over expressed siRNA can affect the regulation of biology network of the body.It might results in the imbalance of proliferation and apoptosis.