ASGPR介导的靶向siRNA抑制HBV的复制与表达

发布时间：2018-04-08 11:40

  本文选题：乙型肝炎病毒　切入点：RNA干扰　出处：《华中科技大学》2009年硕士论文

【摘要】：【目的】 设计与构建针对乙型肝炎病毒（HBV）基因的不同序列特异性siRNA载体，探讨各siRNA对HepG2.2.15细胞HBV复制和表达的作用。筛选获得具有高效沉默HBV的载体，以去唾液酸受体为靶点将其靶向HepG2.2.15细胞，观察靶向与沉默效应、以及对细胞活性的影响，为进一步体内靶向研究奠定基础。 【方法】 1.特异性siRNA真核载体的构建：设计并合成6对针对HBV基因的不同序列siRNA寡核苷酸，，经退火形成双链，然后经T4连接酶连接，克隆入表达EGFP的真核载体，命名为pGenesil-siHBV1～6，并设立与HBV序列无关的pGenesil-siHBV-HK作为阴性对照。 2.重组体的生物活性鉴定：将重组质粒pGenesil-siHBV1～6和pGenesilsiHBV-HK用脂质体分别转染HepG2.2.15细胞。采用实时荧光定量PCR检测转染后2d、3d、4d靶基因的mRNA和细胞培养上清中cccDNA的水平；ELISA检测转染后2d、3d、5d、7d细胞培养上清中HBsAg、HBeAg的表达。 3.去唾液酸受体的靶向作用：将具有高效沉默HBV的siRNA载体利用jetPEI-Hepatocyte靶向转染HepG2.2.15细胞，倒置相差荧光显微镜和流式细胞术（FCM）检测细胞内EGFP的表达，检测转染效率；FCM检测细胞内HBcAg的表达以及转染后3d细胞的凋亡；ELISA检测细胞培养上清中HBsAg、HBeAg的表达，细胞免疫化学酶标技术观察细胞内HBsAg的表达。 【结果】 1. HBV特异性siRNA载体的构建：PCR、酶切及测序鉴定结果表明，针对HBV基因特异性的siRNA真核载体构建成功。 2. HBV特异性siRNA载体的生物活性：Realtime-PCR、ELISA分别从mRNA、蛋白质和cccDNA水平检测，结果表明pGenesil-siHBV1～6均可不同程度的抑制HBV的复制和抗原的表达，其中pGenesil-siHBV1的抑制效果最强。 3. ASGPR靶向pGenesil-siHBV1对HBV的沉默效应：将pGenesil-siHBV1利用jetPEI-Hepatocyte靶向转染HepG2.2.15细胞，倒置相差荧光显微镜下可见绿色荧光，而无jetPEI-Hepatocyte转染对照组未见绿色荧光，FCM检测绿色荧光阳性细胞约为45％；FCM检测细胞内HBcAg的表达以及ELISA检测靶向实验组细胞培养上清中HBsAg、HBeAg的表达低于LipofectamineTM2000转染对照组，且二者均显著低于无关序列对照组；细胞免疫化学酶标技术观察细胞内HBsAg的表达与无关序列对照组比较显著降低；FCM检测转染后3d的细胞凋亡无明显变化。 【结论】 结果表明针对HBV的不同siRNA的沉默效应存在差异；ASGPR介导的靶向递送siRNA载体能更好的发挥沉默HBV基因复制与表达的作用，为下一步的体内实验奠定了良好的基础。
[Abstract]:[purpose]To design and construct different sequence specific siRNA vectors for hepatitis B virus (HBV) gene, and to explore the role of siRNA in HBV replication and expression in HepG2.2.15 cells.The vector with high efficiency silencing HBV was obtained. The sialic acid receptor was used as the target to target HepG2.2.15 cells. The effects of targeting and silencing and the effect on cell activity were observed, which laid a foundation for further research on target in vivo.[methods]1.Construction of specific siRNA eukaryotic vector: six pairs of siRNA oligodeoxynucleotides targeting HBV gene were designed and synthesized. After annealing to form double strand, and then ligated with T4 ligase, they were cloned into eukaryotic vector expressing EGFP.It was named pGenesil-siHBV1 / 6, and pGenesil-siHBV-HK unrelated to HBV sequence was established as negative control.2.Identification of the bioactivity of the recombinant plasmid: the recombinant plasmid pGenesil-siHBV1~6 and pGenesilsiHBV-HK were transfected into HepG2.2.15 cells by liposome respectively.Real-time fluorescence quantitative PCR was used to detect the expression of HBeAg in the cell culture supernatant of 2 days and 3 days after transfection and the level of cccDNA in the supernatant of cell culture by Elisa. The expression of HBeAg was detected in the supernatant of 2 days, 3 days, 5 days and 7 days after transfection.3.The targeting effect of sialic acid receptor: the siRNA vector with high efficiency silencing HBV was transfected into HepG2.2.15 cells by jetPEI-Hepatocyte, and the expression of EGFP was detected by inverted phase contrast fluorescence microscopy and flow cytometry.The expression of HBcAg in cells was detected by FCM and the expression of HBsAg HBeAg in the supernatant of cells was detected by Elisa on the 3rd day after transfection. The expression of HBsAg in cells was observed by immunocytochemical enzyme labeling.[results]1.Construction of HBV specific siRNA vector, restriction endonuclease digestion and sequencing analysis showed that the siRNA eukaryotic vector specific for HBV gene was successfully constructed.2.The biological activity of HBV specific siRNA vector was detected by Elisa from the levels of mRNAs, proteins and cccDNA, respectively. The results showed that pGenesil-siHBV1~6 could inhibit the replication of HBV and the expression of HBV antigens to varying degrees, and pGenesil-siHBV1 had the strongest inhibitory effect.3.The silencing effect of ASGPR targeted pGenesil-siHBV1 on HBV: pGenesil-siHBV1 was transfected into HepG2.2.15 cells by jetPEI-Hepatocyte, and green fluorescence was observed under inverted phase contrast fluorescence microscope.In the control group without jetPEI-Hepatocyte transfection, the expression of HBcAg and the expression of HBeAg in the culture supernatant of targeted experimental group were detected by FCM and the expression of HBeAg in the culture supernatant of targeted experimental group was lower than that in LipofectamineTM2000 transfected control group.The expression of HBsAg in the cells was significantly lower than that in the unrelated sequence control group, and the expression of HBsAg in the cells was significantly lower than that in the unrelated sequence control group on the 3rd day after transfection.[conclusion]The results showed that the silencing effect of different siRNA against HBV was different. ASGPR-mediated targeted delivery siRNA vector could play a better role in silencing the replication and expression of HBV gene, which laid a good foundation for further in vivo experiments.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346

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