本文選題：人巨細胞病毒　切入點：RNA干擾　出處：《山東大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： RNA干擾對HCMV引起平滑肌細胞損傷的保護作用研究 目的 動脈粥樣硬化(Atherosclerosis，AS)嚴重威脅人類健康，是發(fā)達國家人口死亡的主要原因，，在我國AS的發(fā)病率也呈逐年增高的趨勢。研究證明血管炎癥反應(yīng)在動脈粥樣硬化的發(fā)生發(fā)展過程中起關(guān)鍵作用。血清流行病學(xué)和分子生物學(xué)的研究資料表明，人類巨細胞病毒(Human cytomegalovirus，HCMV)抗原及其DNA序列存在于AS的病灶組織，主要見于血管平滑肌細胞(Vascular Smooth Muscle cells，VSMC)和內(nèi)皮細胞(vein endothelial cell，ECV)，且AS患者血清HCMV的抗體陽性率明顯增高，提示HCMV的感染對AS的形成和發(fā)生發(fā)展起重要作用。迄今為止，對于HCMV如何參與AS的發(fā)病機制尚缺乏深入的了解，研究體外情況下HCMV感染后血管壁細胞的生物學(xué)效應(yīng)，對闡明HCMV致AS機理具有非常重要的意義；VSMC表達的氧化型低密度脂蛋白受體-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)最重要的受體之一，也是VSMC結(jié)合或者內(nèi)吞OX-LDL的最重要受體之一，該受體還可誘導(dǎo)單核巨噬細胞轉(zhuǎn)運到血管內(nèi)膜下間隙，刺激平滑肌細胞增生并釋放多種炎性細胞因子，同時VSMC內(nèi)吞OX-LDL轉(zhuǎn)化成泡沫細胞。外周血單核細胞粘附于內(nèi)皮并遷入內(nèi)皮下間隙，攝取脂質(zhì)轉(zhuǎn)化為泡沫細胞是動脈粥樣硬化形成的重要早期事件，單核細胞趨化蛋白-1(monocyte chemoatt ractant protein-1，MCP-1)在這一過程中起重要作用；MCP-1還可通過促進VSMC增殖，參與AS的病理過程。 本課題通過構(gòu)建siRNA質(zhì)粒，調(diào)節(jié)HCMV誘導(dǎo)兔血管平滑肌細胞LOX-1、MCP-1表達以及HCMV的復(fù)制，觀察siRNA質(zhì)粒是否對LOX-1、MCP-1表達以及HCMV復(fù)制有抑制作用，從而明確HCMV通過上調(diào)LOX-1和MCP-1表達參與動脈粥樣硬化發(fā)生發(fā)展的機制，探討siRNA在HCMV感染加劇動脈粥樣硬化發(fā)病機制中的可能治療作用。 方法 1．設(shè)計和構(gòu)建RNA干擾質(zhì)粒 設(shè)計靶基因，采用化學(xué)合成法獲得目的基因，將目的基因ORF與pdsRED2-N1質(zhì)粒連接后成RFP融合蛋白表達載體，進行RNAi有效靶點篩選后用293T細胞進行慢病毒包裝獲得rabLOX-1質(zhì)粒。 2兔主動脈平滑肌細胞培養(yǎng) 將血管中膜的內(nèi)中層進行組織塊培養(yǎng)并鑒定，待細胞呈亞單層狀態(tài)，按常規(guī)方法進行傳代，第3—8代細胞用于實驗。 3基因轉(zhuǎn)染 實驗分為五組：正常細胞對照組：用含2％血清的培養(yǎng)基培養(yǎng)VSMC細胞；rabLOX-1組：按100MOI濃度接種慢病毒質(zhì)粒rabLOX-1，37℃吸附2h后，棄病毒液，用含2％血清的培養(yǎng)基培養(yǎng)細胞；HCMV感染組：以100TCID_(50)／0.1ml濃度接種HCMV AD169病毒株于70-80％融合的VSMC，37℃吸附1h后，棄病毒液，用含2％血清的培養(yǎng)基培養(yǎng)細胞；HCMV+rabLOX-1組：按100TCID_(50)／0.1ml濃度接種HCMVAD169病毒株于VSMC單層細胞，再按100MOI濃度接種慢病毒質(zhì)粒rabLOX-1，用含2％血清的培養(yǎng)基培養(yǎng)細胞；HCMV+空載體組：按100TCID_(50)／0.1ml濃度接種HCMVAD169病毒株于VSMC單層細胞，再按100MOI濃度接種慢病毒空載體，用含2％血清的培養(yǎng)基培養(yǎng)細胞。 4反轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)(RT-PCR)檢測LOX-1mRNA、MCP-1mRNA和HCMVIE72mRNA表達變化 用Trizol提取細胞總RNA，逆轉(zhuǎn)錄合成cDNA，PCR擴增后進行瓊脂糖凝膠電泳，紫外燈下觀察電泳結(jié)果并拍照、掃描。將凝膠電泳圖條帶密度進行計算機分析，計算電泳條帶與GAPDH擴增條帶光密度值作為其相對表達強度。 5蛋白印記法(Western blotting)測LOX-1蛋白表達 收集細胞提取總蛋白并測蛋白濃度后，電泳轉(zhuǎn)膜后，進行免疫反應(yīng)，避光顯色至出現(xiàn)條帶并進行軟件分析。 結(jié)果 1經(jīng)測序及酶切鑒定證明成功構(gòu)建了慢病毒包裝的rabLOX-1； 2免疫組織化學(xué)證實平滑肌細胞原代培養(yǎng)成功； 3 RT-PCR結(jié)果顯示：HCMV+rabLOX-1組中LOX-1mRNA有低水平表達，與正常細胞對照組和rabLOX-1組差異不明顯，在HCMV組和空載體組表達明顯增強(P＜0.01)；MCP-1mRNA在正常細胞對照組和rabLOX-1組表達水平較低，在HCMV組和HCMV+空載體組表達明顯增強，與HCMV+rabLOX-1組、正常細胞對照組及rabLOX-1組均有統(tǒng)計學(xué)意義(P＜0.01)；HCMV IE72mRNA在HCMV組明顯高于HCMV+rabLOX-1組。提示siRNA質(zhì)�？梢杂行б种艸CMV引起的平滑肌細胞LOX-1和MCP-1上調(diào)表達效應(yīng)，抑制病毒自身在細胞內(nèi)的復(fù)制。 4 Western blotting結(jié)果顯示：HCMV組和空載體組LOX-1蛋白與正常對照組、rabLOX-1組和HCMV+rabLOX-1組比較差異有統(tǒng)計學(xué)意義，提示siRNA質(zhì)�？梢杂行б种艸CMV引起的平滑肌細胞LOX-1受體表達效應(yīng)。 結(jié)論 RNA干擾可有效抑制巨細胞病毒誘導(dǎo)的VSMC LOX-1及MCP-1表達上調(diào)，抑制HCMV在細胞內(nèi)的增殖，對VSMC有保護作用。
[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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R373

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