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目的:利用流体动力学法构建小鼠HBV感染模型,观察pSiHBV/P在体内对HBV的抑制作用.方法:以流体动力学法构建小鼠HBV感染模型,将pSiHBV/P与pHBV1.3尾静脉共注射Balb/c小鼠.转染后第6天,用酶联免疫分析法检测小鼠血清乙肝表面抗原(HBsAg),用荧光定量PCR检测血清HBVDNA的水平,用免疫组织化学方法检测肝内HBsAg、乙肝病毒核心抗原(HBcAg)的表达,RT-PCR法半定量检测肝内HBV3.5kb mRNA、S-mRNA及0.7kb mRNA的水平.结果:感染组和无关干扰组血清HBsAg表达量平均值分别为9.11±3.34和8.19±5.41,pSiHBV/P干扰组平均值1.94±1.78;pSiHBV/P干扰组血清中HBV DNA的含量较感染组明显下降(2.91±0.55vs4.32±0.57,P<0.05),抑制率为33%;干扰组肝细胞中HBsAg和HBcAg阳性细胞数较对照组明显减少;干扰组3.5kb mRNA、S-mRNA的水平较感染组和无关干扰组均有明显下降(0.48±0.19vs1.06±0.40,0.88±0.54;0.46±0.18vs1.05±0.38,0.90±0.51,P<0.05).结论:RNAi技术在体内能高效、特异地抑制小鼠体内HBV.
OBJECTIVE: To establish a murine model of HBV infection by hydrodynamic method and observe the inhibitory effect of pSiHBV / P on HBV in vivo.Methods: The murine model of HBV infection was established by hydrodynamic method, and pSiHBV / P was mixed with pHBV1.3 tail vein Balb / c mice were co-injected on the 6th day after transfection.Serum HBsAg was detected by enzyme-linked immunosorbent assay (ELISA), HBVDNA level was detected by real-time quantitative PCR and immunohistochemistry HBsAg and HBcAg were detected by RT-PCR, and the levels of HBV 3.5 kb mRNA, S-mRNA and 0.7 kb mRNA in the liver were detected by RT-PCR.Results: The average level of HBsAg in the infected group and non-related group (9.11 ± 3.34 and 8.19 ± 5.41 respectively), and the mean value of pSiHBV / P interference group was 1.94 ± 1.78. The serum HBV DNA levels of pSiHBV / P interference group were significantly lower than those of infected group (2.91 ± 0.55 vs 4.32 ± 0.57, P <0.05) ), And the inhibition rate was 33%. The number of HBsAg and HBcAg positive cells in the interference group was significantly lower than that in the control group; the expression of 3.5kb mRNA and S-mRNA in the interference group was significantly lower than that in the infection group and the unrelated interference group (0.48 ± 0.19vs1.06 ± 0.40,0.88 ± 0.54; 0.46 ± 0.18vs1.05 ± 0.38,0.90 ± 0.51, P <0.05) .Conclusion: RNAi Technology in the body can efficiently and specifically inhibit HBV in mice.