论文部分内容阅读
目的建立小鼠急性乙型肝炎病毒感染的动物模型,在此基础上观察小干扰RNA(siRNA)对HBV复制和表达的影响。方法通过尾静脉注射1.3倍的HBV真核表达质粒pHBV1.3建立小鼠急性乙型肝炎感染模型,再将其与针对乙型肝炎病毒核心区的siRNA表达载体(pSI C)共注射,于注射后第6天取血测血清HBsAg(ELISA),做肝组织HBcAg免疫组化,通过RT PCR检测肝组织HBVC区mRNA转录子的水平。同时设立PBS对照组、无关干扰组与突变干扰组。结果转染后第6天血清HBSAg在pHBV1.3感染组中高表达(A值为4.08～9.04,平均值为5.07±1.07,A值≥2.1为阳性),肝内HBcAg阳性率为5%～10%;pSI C干扰组HBSAg,HBcAg的表达均受到抑制,血清HBsAg阴性,A值为0.04～1.5,平均值为0.62±0.59,与感染组相比有显著性差异(P<0.01),肝内HBcAg几乎无表达,RT PCR示肝内HBVCmRNA水平明显降低。而无关干扰pGFP及突变的干扰序列pSI Cmut则无此作用。结论RNAi技术能特异,高效地抑制小鼠体内HBV的复制和表达,提示siRNA作为一种新型抗病毒药物的巨大潜能,而通过水动力法建立的小鼠急性乙型肝炎病毒感染的动物模型,一定程度上解决了长期以来缺乏合适的HBV感染动物模型的问题,为今后进行药物筛选及抗病毒治疗的研究拓展了新的思路。
Objective To establish an animal model of acute hepatitis B virus infection in mice and observe the effect of siRNA on HBV replication and expression. Methods A mouse model of acute hepatitis B infection was established by injecting a 1.3-fold HBV DNA eukaryotic expression plasmid pHBV1.3 into the caudal vein and then co-injected with the siRNA expression vector (pSI C) against the core of hepatitis B virus. Six days later, blood was taken for serum HBsAg (ELISA), and immunohistochemistry of HBcAg in liver tissue was performed. The level of mRNA transcript in HBVC region of liver tissue was detected by RT-PCR. Meanwhile, PBS control group, irrelevant interference group and mutation interference group were set up. Results On the 6th day after transfection, the serum HBsAg was highly expressed in the group of pHBV1.3 infection (A value 4.08 ~ 9.04, mean 5.07 ± 1.07, A value ≥2.1 positive). The positive rate of HBcAg in the liver was 5% ~ 10 %; The expression of HBsAg and HBcAg in the group of pSI C was inhibited, the serum HBsAg was negative, the value of A was 0.04-1.5, the average was 0.62 ± 0.59, which was significantly different from that of the infected group (P <0.01) HBcAg almost no expression, RT PCR showed significant reduction in intrahepatic HBVC mRNA levels. However, no interference with pGFP and mutation of the interference sequence pSI Cmut no such role. Conclusion RNAi can specifically and efficiently inhibit the replication and expression of HBV in mice, suggesting the huge potential of siRNA as a novel antiviral drug. However, the animal model of acute hepatitis B virus infection in mice established by hydrodynamic method, To a certain extent, it solves the problem of lacking a suitable animal model of HBV infection for a long time and expands the new thinking for future research on drug screening and antiviral therapy.