小鼠B7-DC基因沉默对树突状细胞诱导的抗乙肝病毒免疫的研究

发布时间：2018-01-19 11:09

本文关键词： 乙肝病毒原核表达树突状细胞 B7-DC RNA干扰　出处：《华东师范大学》2009年硕士论文　论文类型：学位论文

【摘要】： 乙型肝炎病毒是一种非细胞裂解性病毒,它是引起急、慢性肝炎的主要病原。研究发现,在慢性乙肝患者体内的CD8+ T细胞的增殖和效应能力很弱,提示了增强病毒特异性T细胞的反应能力可能是控制慢性乙肝病毒持续感染的一条途径。目前,树突状细胞疫苗被公认为是抗肿瘤和抗慢性病毒感染的有效方法之一。因此,基于树突状细胞的治疗性疫苗对于乙肝的免疫治疗是可行的。许多研究表明,B7家族提供的共刺激信号对促进和抑制T细胞活化具有至关重要的作用。B7-DC是B7家族的第五个成员,被认为是PD-1的第二个配体,可以负向调节T细胞的活化。B7-DC选择性表达于树突状细胞上,因此,如果阻断树突状细胞与T细胞B7-DC/PD-1间的相互作用,有可能使树突状细胞诱导更强的T细胞反应。 RNA干扰是一种新颖的基因调节机制,是通过双链RNA介导的转录后基因沉默技术,它是目前封闭基因表达行之有效的方法之一。本研究中,我们从小鼠骨髓来源的未成熟树突状细胞中提取总RNA,经RT-PCR扩增B7-DC胞外段,并将其克隆至原核表达载体pET32a(+)中,构建重组表达质粒pET32a(+)-B7-DC_(ECD)。重组质粒经双酶切鉴定及序列测定后,转化E.coliBL21,经IPTG诱导表达目的蛋白,并用SDS-PAGE和Western blot进行检测。实验结果表明,成功获得全长为582 bp的小鼠B7-DC胞外段基因,经测序证实其序列正确。SDS-PAGE和Western blot分析证实重组质粒可表达出Mr约为41 000的B7-DC胞外段蛋白。同时我们对携带有针对目标基因的shRNA在抑制树突状细胞上B7-DC基因的表达水平,以及基因修饰的树突状细胞的功能方面作了初步的研究。成功构建两个siRNA真核表达载体并在体外转染树突状细胞,检测结果表明两个siRNA真核表达载体均可抑制树突状细胞上B7-DC基因的表达,重组干扰质粒shRNA1-B7-DC和shRNA2-B7-DC抑制B7-DC基因的效率分别为61.4%、45.9%。混合淋巴细胞反应中B7-DC基因沉默的树突状细胞可显著刺激同种异体T淋巴细胞增殖。将基因修饰的树突状细胞免疫HBV转基因小鼠,检测结果表明在效:靶比为50:1时,经shRNA1-B7-DC转染后致敏的DCs细胞免疫组小鼠脾细胞CTL杀伤活性显著高于空载体pAS转染组和非转染组,提示树突状细胞中B7-DC基因沉默后诱导更强的CTL杀伤活性。此外,我们检测了免疫小鼠血清中乙肝表面抗原的水平和血清中HBV DNA的浓度。结果显示,乙肝病毒特异性表位肽脉冲的树突状细胞可诱导特异性的抗乙肝病毒免疫,显著降低了血清中乙肝表面抗原和HBV DNA的浓度,提示了B7-DC基因沉默在树突状细胞疫苗治疗乙肝上的潜在应用价值。
[Abstract]:Hepatitis B virus is a non-cellular lytic virus, it is the main cause of acute and chronic hepatitis. The study found that the proliferation and effect of CD8 T cells in patients with chronic hepatitis B is very weak. It is suggested that enhancing the response ability of viral specific T cells may be a way to control the persistent infection of chronic hepatitis B virus. Dendritic cell vaccine is recognized as one of the effective methods of anti-tumor and anti-chronic virus infection. Therefore, the therapeutic vaccine based on dendritic cells is feasible for hepatitis B immunotherapy. Many studies have shown that the costimulatory signal provided by the B7 family plays an important role in promoting and inhibiting the activation of T cells. B7-DC is the fifth member of the B7 family. It is considered to be the second ligand of PD-1, which can negatively regulate the activation of T cells. B7-DC is selectively expressed on dendritic cells. If the interaction between dendritic cells and T cells B7-DC-PD-1 is blocked, it is possible that dendritic cells induce stronger T cell responses. RNA interference is a novel gene regulation mechanism, which is a post-transcriptional gene silencing technique mediated by double-stranded RNA. It is one of the effective methods for blocking gene expression. In this study, we extracted total RNAs from immature dendritic cells from mouse bone marrow and amplified the extracellular segments of B7-DC by RT-PCR. The recombinant plasmid pET32a was cloned into prokaryotic expression vector pET32a (pET32a), and the recombinant plasmid was identified by double enzyme digestion and sequenced. E. coli BL21 was transformed into E. coli BL21. The target protein was induced by IPTG and detected by SDS-PAGE and Western blot. A 582 BP mouse B7-DC extracellular segment gene was successfully obtained. Sequencing confirmed that its sequence was correct. SDS-PAGE and Western blot analysis confirmed that the recombinant plasmid could express Mr 41. At the same time, we inhibited the expression of B7-DC gene in dendritic cells by shRNA carrying target gene. Two siRNA eukaryotic expression vectors were successfully constructed and transfected into dendritic cells in vitro. The results showed that two siRNA eukaryotic expression vectors could inhibit the expression of B7-DC gene in dendritic cells. The efficiency of inhibition of B7-DC gene by recombinant interference plasmid shRNA1-B7-DC and shRNA2-B7-DC was 61.4%, respectively. The dendritic cells silenced by B7-DC gene could significantly stimulate the proliferation of allogeneic T lymphocytes. The genetically modified dendritic cells were immunized with HBV transgenic mice. The results show that the target ratio is 50: 1. The CTL killing activity of spleen cells in DCs cells immunized with shRNA1-B7-DC was significantly higher than that in empty vector pAS transfection and non-transfection groups. These results suggest that B7-DC gene silencing induces stronger CTL killing activity in dendritic cells. The serum level of hepatitis B surface antigen and the concentration of HBV DNA in serum of immunized mice were determined. Dendritic cells with HBV-specific epitope peptide pulse could induce specific anti-HBV immunity and significantly reduce the concentration of HBV surface antigen and HBV DNA in serum. The potential value of B 7-DC gene silencing in the treatment of hepatitis B with dendritic cell vaccine was suggested.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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