RNA干扰对HCMV引起平滑肌细胞损伤的保护作用研究

发布时间：2018-03-10 01:30

本文选题：人巨细胞病毒　切入点：RNA干扰　出处：《山东大学》2007年硕士论文　论文类型：学位论文

【摘要】： RNA干扰对HCMV引起平滑肌细胞损伤的保护作用研究目的动脉粥样硬化(Atherosclerosis，AS)严重威胁人类健康，是发达国家人口死亡的主要原因，，在我国AS的发病率也呈逐年增高的趋势。研究证明血管炎症反应在动脉粥样硬化的发生发展过程中起关键作用。血清流行病学和分子生物学的研究资料表明，人类巨细胞病毒(Human cytomegalovirus，HCMV)抗原及其DNA序列存在于AS的病灶组织，主要见于血管平滑肌细胞(Vascular Smooth Muscle cells，VSMC)和内皮细胞(vein endothelial cell，ECV)，且AS患者血清HCMV的抗体阳性率明显增高，提示HCMV的感染对AS的形成和发生发展起重要作用。迄今为止，对于HCMV如何参与AS的发病机制尚缺乏深入的了解，研究体外情况下HCMV感染后血管壁细胞的生物学效应，对阐明HCMV致AS机理具有非常重要的意义；VSMC表达的氧化型低密度脂蛋白受体-1(Lectin-like oxidized low density lipoprotein receptor-1，LOX-1)是氧化型低密度脂蛋白(oxidized low density lipoprotein，OX-LDL)最重要的受体之一，也是VSMC结合或者内吞OX-LDL的最重要受体之一，该受体还可诱导单核巨噬细胞转运到血管内膜下间隙，刺激平滑肌细胞增生并释放多种炎性细胞因子，同时VSMC内吞OX-LDL转化成泡沫细胞。外周血单核细胞粘附于内皮并迁入内皮下间隙，摄取脂质转化为泡沫细胞是动脉粥样硬化形成的重要早期事件，单核细胞趋化蛋白-1(monocyte chemoatt ractant protein-1，MCP-1)在这一过程中起重要作用；MCP-1还可通过促进VSMC增殖，参与AS的病理过程。本课题通过构建siRNA质粒，调节HCMV诱导兔血管平滑肌细胞LOX-1、MCP-1表达以及HCMV的复制，观察siRNA质粒是否对LOX-1、MCP-1表达以及HCMV复制有抑制作用，从而明确HCMV通过上调LOX-1和MCP-1表达参与动脉粥样硬化发生发展的机制，探讨siRNA在HCMV感染加剧动脉粥样硬化发病机制中的可能治疗作用。方法 1．设计和构建RNA干扰质粒设计靶基因，采用化学合成法获得目的基因，将目的基因ORF与pdsRED2-N1质粒连接后成RFP融合蛋白表达载体，进行RNAi有效靶点筛选后用293T细胞进行慢病毒包装获得rabLOX-1质粒。 2兔主动脉平滑肌细胞培养将血管中膜的内中层进行组织块培养并鉴定，待细胞呈亚单层状态，按常规方法进行传代，第3—8代细胞用于实验。 3基因转染实验分为五组：正常细胞对照组：用含2％血清的培养基培养VSMC细胞；rabLOX-1组：按100MOI浓度接种慢病毒质粒rabLOX-1，37℃吸附2h后，弃病毒液，用含2％血清的培养基培养细胞；HCMV感染组：以100TCID_(50)／0.1ml浓度接种HCMV AD169病毒株于70-80％融合的VSMC，37℃吸附1h后，弃病毒液，用含2％血清的培养基培养细胞；HCMV+rabLOX-1组：按100TCID_(50)／0.1ml浓度接种HCMVAD169病毒株于VSMC单层细胞，再按100MOI浓度接种慢病毒质粒rabLOX-1，用含2％血清的培养基培养细胞；HCMV+空载体组：按100TCID_(50)／0.1ml浓度接种HCMVAD169病毒株于VSMC单层细胞，再按100MOI浓度接种慢病毒空载体，用含2％血清的培养基培养细胞。 4反转录聚合酶链式反应(RT-PCR)检测LOX-1mRNA、MCP-1mRNA和HCMVIE72mRNA表达变化用Trizol提取细胞总RNA，逆转录合成cDNA，PCR扩增后进行琼脂糖凝胶电泳，紫外灯下观察电泳结果并拍照、扫描。将凝胶电泳图条带密度进行计算机分析，计算电泳条带与GAPDH扩增条带光密度值作为其相对表达强度。 5蛋白印记法(Western blotting)测LOX-1蛋白表达收集细胞提取总蛋白并测蛋白浓度后，电泳转膜后，进行免疫反应，避光显色至出现条带并进行软件分析。结果 1经测序及酶切鉴定证明成功构建了慢病毒包装的rabLOX-1； 2免疫组织化学证实平滑肌细胞原代培养成功； 3 RT-PCR结果显示：HCMV+rabLOX-1组中LOX-1mRNA有低水平表达，与正常细胞对照组和rabLOX-1组差异不明显，在HCMV组和空载体组表达明显增强(P＜0.01)；MCP-1mRNA在正常细胞对照组和rabLOX-1组表达水平较低，在HCMV组和HCMV+空载体组表达明显增强，与HCMV+rabLOX-1组、正常细胞对照组及rabLOX-1组均有统计学意义(P＜0.01)；HCMV IE72mRNA在HCMV组明显高于HCMV+rabLOX-1组。提示siRNA质粒可以有效抑制HCMV引起的平滑肌细胞LOX-1和MCP-1上调表达效应，抑制病毒自身在细胞内的复制。 4 Western blotting结果显示：HCMV组和空载体组LOX-1蛋白与正常对照组、rabLOX-1组和HCMV+rabLOX-1组比较差异有统计学意义，提示siRNA质粒可以有效抑制HCMV引起的平滑肌细胞LOX-1受体表达效应。结论 RNA干扰可有效抑制巨细胞病毒诱导的VSMC LOX-1及MCP-1表达上调，抑制HCMV在细胞内的增殖，对VSMC有保护作用。
[Abstract]:Protective effect of RNA interference on smooth muscle cell injury induced by HCMV
objective
Atherosclerosis (Atherosclerosis, AS) a serious threat to human health, is the main cause of death in developed countries, in the incidence of AS was also increased year by year. Studies show that vascular inflammation plays a key role in the initiation and progression of atherosclerosis. The serum epidemiology and molecular biology research data show that human cytomegalovirus (Human cytomegalovirus HCMV) antigen and DNA sequence in AS lesions, mainly in vascular smooth muscle cells (Vascular Smooth Muscle cells, VSMC) and endothelial cells (vein endothelial cell, ECV), and the positive rate of AS antibody in serum of patients with HCMV significantly increased, suggesting that HCMV infection of the AS formation and occurrence played an important role in the development. So far, the pathogenesis of HCMV is how to participate in AS's lack of understanding, after the case study of in vitro vascular HCMV infection The biological effects of cell wall, is very important to clarify the mechanism of HCMV induced by AS has the significance of the expression of VSMC; oxidized low density lipoprotein receptor -1 (Lectin-like oxidized low density lipoprotein receptor-1, LOX-1) is oxidized low density lipoprotein (oxidized low density lipoprotein, OX-LDL) is one of the most important receptor, one of the most important receptor or endocytosis of OX-LDL VSMC is the combination of the receptor can be induced by macrophages to transport vascular intima, stimulate the proliferation of smooth muscle cells and inflammatory cytokines, VSMC and endocytosis of OX-LDL into foam cells. Peripheral blood mononuclear cells adhesion to endothelial and subendothelial space in lipid uptake, transformation foam cells is an important early event in atherogenesis, monocyte chemoattractant protein -1 (monocyte chemoatt ractant protein-1, MCP-1) in this. It plays an important role in the process, and MCP-1 can also participate in the pathological process of AS by promoting the proliferation of VSMC.
This topic through the construction of siRNA plasmid, the regulation of HCMV induced rabbit vascular smooth muscle cells LOX-1, MCP-1 expression and HCMV replication, to observe whether siRNA plasmid of LOX-1, expression of MCP-1 and inhibit the replication of HCMV, so as to clear the HCMV by up regulating LOX-1 and MCP-1 expression is involved in the mechanism of atherosclerosis occurrence and development, explore the role of siRNA in HCMV infection treatment the role in the pathogenesis of atherosclerosis increased.
Method
1. design and construct RNA interference plasmids
The target gene was designed, and the target gene was obtained by chemical synthesis. After connecting the target gene ORF with pdsRED2-N1 plasmid, the expression vector of RFP fusion protein was obtained. RNAi effective target was screened, and rabLOX-1 plasmid was packaged by lentivirus packaged with 293T cells.
2 rabbit aortic smooth muscle cell culture
The medial middle layer of the middle membrane of the vessel was cultured and identified. The cells were subcultured in a submonolayer state. The third to 8 generation cells were used for the experiment.
3 gene transfection
The experiment was divided into five groups: control group of normal cells: VSMC cells were cultured in medium containing 2% serum; rabLOX-1 group: according to the concentration of 100MOI inoculated with rabLOX-1,37 lentivirus vector C after 2H adsorption, abandon the virus liquid cell culture medium containing 2% serum; group HCMV infection: 100TCID_ (50) / 0.1ml concentration inoculation of HCMV AD169 strains in 70-80% fusion VSMC, 37 DEG C after 1h adsorption, abandon the virus liquid cell culture medium containing 2% serum; group HCMV+rabLOX-1: 100TCID_ (50) 0.1ml / HCMVAD169 strains in VSMC were inoculated monolayer cells, and then 100MOI concentration inoculated with plasmid rabLOX-1, the cultured cells with medium containing 2% serum; HCMV+ vector group: according to 100TCID_ (50) 0.1ml / HCMVAD169 strains in VSMC were inoculated monolayer cells, according to the concentration of 100MOI inoculated with lentiviral vector, using culture medium containing 2% serum fine Cell.
4 reverse transcriptional polymerase chain reaction (RT-PCR) detection of LOX-1mRNA, MCP-1mRNA and HCMVIE72mRNA expression
Total cellular RNA was extracted by Trizol, cDNA was synthesized, amplified by PCR and agarose gel electrophoresis, UV light and photographed agarose gel electrophoresis, scanning. The band densities were analyzed by computer calculation, electrophoresis and GAPDH amplification band optical density value as its relative expression intensity.
The expression of LOX-1 protein by 5 protein imprinting (Western blotting)
After collecting the total protein and measuring the protein concentration, the immune reaction was carried out after the electrophoretic conversion was carried out, and the color appeared to appear and the software was analyzed.
Result
1 the rabLOX-1 was successfully constructed by sequencing and identification of the lentivirus.
2 immuno histochemistry proved that the primary culture of smooth muscle cells was successful.
3 RT-PCR results showed that LOX-1mRNA HCMV+rabLOX-1 group had lower levels, and the normal control group and rabLOX-1 group were not significantly different, the expression increased significantly in HCMV group and empty vector group (P < 0.01); MCP-1mRNA control group and rabLOX-1 group in normal cells lower expression, expression was significantly increased in group HCMV and HCMV+ the empty vector group, and HCMV+rabLOX-1 group, normal control group and rabLOX-1 group were statistically significant (P < 0.01); HCMV IE72mRNA in HCMV group was significantly higher than that in HCMV+rabLOX-1 group. SiRNA plasmid can effectively inhibit HCMV induced smooth muscle cells LOX-1 and MCP-1 expression effect, its inhibition of virus replication in cells.
4 Western blotting results showed that there was a significant difference between the LOX-1 protein of HCMV group and the empty carrier group compared with the normal control group, rabLOX-1 group and HCMV+rabLOX-1 group, suggesting that siRNA plasmid can effectively inhibit HCMV induced expression of LOX-1 receptor in smooth muscle cells.
conclusion
RNA interference can effectively inhibit the up-regulated expression of VSMC LOX-1 and MCP-1 induced by cytomegalovirus, inhibit the proliferation of HCMV in cell and protect VSMC.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R373

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