本文關(guān)鍵詞： 重組腺病毒 分化抑制因子1 肝癌 乙型肝炎病毒 動(dòng)物模型　出處：《重慶醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:構(gòu)建分化抑制因子1(Id1)重組腺病毒(Rad),檢測(cè)RAd-Id1在細(xì)胞學(xué)水平和小鼠體內(nèi)的表達(dá)水平及其引起的相應(yīng)變化,初步研究肝癌細(xì)胞中Id1的表達(dá)對(duì)乙型肝炎病毒復(fù)制水平的影響。方法:先將Id1基因克隆到pAdtrack-TO4上,再重組到Ad Easy-1腺病毒骨架載體中;在HEK293細(xì)胞中采用Tong-Chuan He[1]等方法將腺病毒重組質(zhì)粒pAdEasy-Id1進(jìn)行包裝以獲得Id1重組腺病毒(RAd-Id1),通過綠色熒光蛋白標(biāo)記法測(cè)定腺病毒滴度;通過qRT-PCR和Western blot實(shí)驗(yàn)驗(yàn)證RAd-Id1感染的Hep G2細(xì)胞中Id1的過表達(dá)效果;用MTS比色法檢測(cè)RAd-Id1對(duì)HepG2細(xì)胞增殖的影響;通過尾靜脈注射RAd-Id1的感染方式建立肝組織中過表達(dá)Id1的小鼠動(dòng)物模型,病毒載量梯度實(shí)驗(yàn)分PBS空白組、RAd-GFP對(duì)照組及RAd-Id1實(shí)驗(yàn)組(n=5/組)以測(cè)定RAd-Id1感染小鼠的適合載量;病毒感染時(shí)間梯度實(shí)驗(yàn)每5天檢測(cè)一次為一組,共7組(n=5/組)以測(cè)定感染小鼠的適合時(shí)間;通過Western blot檢測(cè)小鼠肝組織中Id1的過表達(dá)效果及甲胎蛋白(AFP)和癌胚抗原(CEA)的表達(dá)水平,再采用ELISA檢測(cè)小鼠血清中Id1的免疫原性及AFP和CEA的表達(dá)情況;RAd-Id1感染穩(wěn)定表達(dá)HBV的肝癌細(xì)胞系HepG2.2.15,觀察HBV復(fù)制水平的變化。結(jié)果:成功構(gòu)建Id1-p Adtrack-TO4和Id1-p AdEasy-1質(zhì)粒,并包裝獲得RAd-Id1,測(cè)得其滴度為1.5×1011GFU/mL;RAd-Id1感染HepG2細(xì)胞48 h和72 h后,Id1的轉(zhuǎn)錄和蛋白水平均明顯升高(P0.01);96 h時(shí)RAd-Id1實(shí)驗(yàn)組的MTS檢測(cè)值較PBS空白組和RAd-GFP對(duì)照組明顯升高;WB結(jié)果顯示:在RAd-Id1感染的小鼠肝組織中,Id1、AFP和CEA蛋白水平較PBS空白組和RAd-GFP對(duì)照組升高;Hep G2.2.15細(xì)胞在感染RAd-Id1后HBV的復(fù)制(HBV DNA)和轉(zhuǎn)錄(HBV pgRNA和HBc蛋白)水平降低。結(jié)論:成功構(gòu)建了RAd-Id1及其介導(dǎo)的小鼠動(dòng)物模型;RAd-Id1可作為細(xì)胞水平上Id1對(duì)肝癌和HBV作用機(jī)制研究的載體工具;AFP和CEA的升高提示Id1可能參與誘導(dǎo)AFP和CEA的表達(dá);Id1可能參與抑制HBV復(fù)制和表達(dá)水平的過程。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.7;R512.62

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