分化抑制因子1重组腺病毒（RAd-Id1）的构建及其相关功能的研究

发布时间：2018-02-27 23:03

本文关键词： 重组腺病毒分化抑制因子1 肝癌乙型肝炎病毒动物模型　出处：《重庆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:构建分化抑制因子1(Id1)重组腺病毒(Rad),检测RAd-Id1在细胞学水平和小鼠体内的表达水平及其引起的相应变化,初步研究肝癌细胞中Id1的表达对乙型肝炎病毒复制水平的影响。方法:先将Id1基因克隆到pAdtrack-TO4上,再重组到Ad Easy-1腺病毒骨架载体中;在HEK293细胞中采用Tong-Chuan He[1]等方法将腺病毒重组质粒pAdEasy-Id1进行包装以获得Id1重组腺病毒(RAd-Id1),通过绿色荧光蛋白标记法测定腺病毒滴度;通过qRT-PCR和Western blot实验验证RAd-Id1感染的Hep G2细胞中Id1的过表达效果;用MTS比色法检测RAd-Id1对HepG2细胞增殖的影响;通过尾静脉注射RAd-Id1的感染方式建立肝组织中过表达Id1的小鼠动物模型,病毒载量梯度实验分PBS空白组、RAd-GFP对照组及RAd-Id1实验组(n=5/组)以测定RAd-Id1感染小鼠的适合载量;病毒感染时间梯度实验每5天检测一次为一组,共7组(n=5/组)以测定感染小鼠的适合时间;通过Western blot检测小鼠肝组织中Id1的过表达效果及甲胎蛋白(AFP)和癌胚抗原(CEA)的表达水平,再采用ELISA检测小鼠血清中Id1的免疫原性及AFP和CEA的表达情况;RAd-Id1感染稳定表达HBV的肝癌细胞系HepG2.2.15,观察HBV复制水平的变化。结果:成功构建Id1-p Adtrack-TO4和Id1-p AdEasy-1质粒,并包装获得RAd-Id1,测得其滴度为1.5×1011GFU/mL;RAd-Id1感染HepG2细胞48 h和72 h后,Id1的转录和蛋白水平均明显升高(P0.01);96 h时RAd-Id1实验组的MTS检测值较PBS空白组和RAd-GFP对照组明显升高;WB结果显示:在RAd-Id1感染的小鼠肝组织中,Id1、AFP和CEA蛋白水平较PBS空白组和RAd-GFP对照组升高;Hep G2.2.15细胞在感染RAd-Id1后HBV的复制(HBV DNA)和转录(HBV pgRNA和HBc蛋白)水平降低。结论:成功构建了RAd-Id1及其介导的小鼠动物模型;RAd-Id1可作为细胞水平上Id1对肝癌和HBV作用机制研究的载体工具;AFP和CEA的升高提示Id1可能参与诱导AFP和CEA的表达;Id1可能参与抑制HBV复制和表达水平的过程。
[Abstract]:Objective: to construct Recombinant adenovirus Radon of differentiation inhibitor 1 (ID1), and to detect the expression of RAd-Id1 at the cytological level and the expression level in mice and its corresponding changes. Methods: Id1 gene was cloned into pAdtrack-TO4 and then recombined into Ad Easy-1 adenovirus skeleton vector. Adenovirus recombinant plasmid pAdEasy-Id1 was packaged in HEK293 cells by Tong-Chuan he [1] to obtain Id1 recombinant adenovirus, RAd-Id1, and the titer of adenovirus was determined by green fluorescent protein labeling method. QRT-PCR and Western blot experiments were used to verify the effect of Id1 overexpression in HepG2 cells infected with RAd-Id1, MTS colorimetric assay was used to detect the effect of RAd-Id1 on the proliferation of HepG2 cells, and a mouse model of overexpression of Id1 in liver tissue was established by injecting RAd-Id1 via tail vein. The virus load gradient test was divided into PBS blank group (RAd-GFP control group) and RAd-Id1 experimental group (RAd-GFP control group) to determine the suitable load of RAd-Id1 infected mice, and the virus infection time gradient test was performed every 5 days as a group. Western blot was used to detect the expression of Id1 and carcinoembryonic antigen (CEA) in liver tissue of 7 groups. ELISA was used to detect the immunogenicity of Id1 and the expression of AFP and CEA in mouse serum. RAd-Id1 infected HepG2.2.15 hepatoma cell line with stable expression of HBV. Results: Id1-p Adtrack-TO4 and Id1-p AdEasy-1 plasmids were constructed successfully. RAd-Id1 was obtained by packaging. The titer of RAd-Id1 was 1.5 脳 10 ~ (11) GFU / m ~ (-1) RAd-Id1 in HepG2 cells infected 48 h and 72 h later, the transcription and protein levels of RAd-Id1 were significantly increased at 96 h after infection with RAd-Id1. The MTS detection value of RAd-Id1 experimental group was significantly higher than that of PBS control group and RAd-GFP control group. Compared with PBS control group and RAd-GFP control group, the levels of CEA and AFP protein in RAd-Id1 infected mice liver tissue increased significantly the levels of HBV replication HBV DNA and HBV pgRNA and HBc protein in Hep G2.2.15 cells after RAd-Id1 infection. Conclusion: RAd-Id1 and HBc protein were successfully constructed. RAd-Id1 mediated by RAd-Id1 can be used as a carrier tool to study the mechanism of Id1 on hepatocarcinoma and HBV. The increase of Id1 and CEA suggests that Id1 may be involved in the process of inducing AFP and CEA to express AFP and CEA and inhibit HBV replication and expression level.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7;R512.62

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