【作者】 王玮；

【导师】 刘运海；

【作者基本信息】 中南大学 ， 神经病学， 2010， 博士

【摘要】 目前,脑卒中(stroke)是严重危害人类健康的常见病、多发病,具有发病率高、致残率高及死亡率高的特点。其中脑梗死占全部脑卒中的60%-80%,而动脉粥样硬化(atherosclerosis, AS)是引起脑梗死的最主要原因的之一。动脉粥样硬化斑块表面的纤维帽或是内膜损伤导致斑块破裂从而发生严重的并发症如脑梗死等。因此对AS发病机理进行研究并对高危人群采取适当的干预措施,将有助于减少动脉粥样硬化性脑梗死的发生。近几十年来人们致力于研究动脉粥样硬化的发病机制,已经发现其与炎症、高血脂等多种因素相关,但其具体作用机制仍不甚明确。血管壁由内皮细胞、平滑肌细胞以及基质等组成,这些成分组成一个统一体,但若要具有相应的功能则需相互间进行化学和电信号的交换。在这些信息通讯通路中,一个重要途径就是缝隙连接通讯。缝隙连接(gap junction, GJ)是缝隙连接通讯的结构基础,是由缝隙连接蛋白(Connexin, Cx)组成的细胞膜通道,允许离子、小的分子物质以及第二信使在两个相邻细胞间进行交换。近年来人们发现缝隙连接与AS密切相关,但其作用机制尚不清楚。另外,最近对缝隙连接蛋白37(Connexin 37, Cx37)基因多态与AS及冠心病(coronary heart disease, CHD)、心肌梗死(myocardial infarction, MI)之间进行了大量相关性研究,发现Cx37 C1019T位点是AS的“单基因标志物”,与冠心病、心肌梗死密切相关。而与冠心病和心肌梗死一样以动脉粥样硬化性为病理基础的脑梗死是否与Cx37基因多态相关?目前国内外尚未见报道。因此我们通过观察氧化型低密度脂蛋白(oxidized low-density lipoprotein, ox-LDL)、高密度脂蛋白(high-densitylipoprotein, HDL)和缝隙连接抑制剂辛醇(octanol)对人脐静脉内皮细胞缝隙连接蛋白表达的影响,从细胞水平研究缝隙连接与AS的关系,并进一步探讨其中的机制。另外还在基因水平研究了Cx37基因多态和动脉粥样硬化性脑梗死的相关性。目的：体外培养人脐静脉内皮细胞株HUVE-12,用ox-LDL诱导内皮细胞损伤,建立动脉粥样硬化损伤内皮细胞模型,观察其对内皮细胞缝隙连接蛋白表达、细胞凋亡和线粒体凋亡途径的影响,同时用HDL或octanol和ox-LDL共孵育对内皮细胞Cx表达和细胞凋亡的影响并探讨其机制。方法：无菌培养HUVE-12,实验分为7组：①对照组：普通DMEM培养基；②-④ox-LDL组：培养基中分别加入终浓度为25μg/ml、50μg/ml和100μg/ml ox-LDL孵育24小时；⑤HDL组：培养基中加入终浓度为100μg/mlHDL孵育24小时；⑥HDL+ox-LDL组：培养基中加入终浓度为100μg/mlHDL和50μg/ml ox-LDL共同孵育24小时；⑦octanol+ox-LDL组：培养基中加入终浓度为1mM octanol和50μg/ml ox-LDL共同孵育24小时。分别用免疫细胞化学方法(Immunocytochemistry, ICC)、western blot方法和实时荧光定量RT-PCR (real-time fluorescent quantitative RT-PCR)检测各组细胞缝隙连接蛋白Cx37、Cx40和Cx43以及Bcl-2、Bax和Caspase-3蛋白和相应mRNA的表达；MTT方法检测细胞增殖,台盼蓝拒染法检测细胞活性和存活率,流式细胞仪检测细胞凋亡。结果：1、与对照组比较,25μg／ml、50μg／ml和100μg／ml ox-LDL组内皮细胞上缝隙连接蛋白Cx37、Cx40蛋白和mRNA的表达水平下调,Cx43蛋白和mRNA表达水平上调,其差异均具有统计学意义(P<0.05)。同时25μg／ml、50μg／ml和100μg／ml ox-LDL组细胞增殖受到明显抑制,细胞凋亡率显著增加,细胞存活率下降,与对照组比较,其差异均具有统计学意义(P<0.05)。25μg／ml、50μg／ml和100μg／ml ox-LDL组线粒体凋亡途径的抑凋亡基因Bcl-2蛋白和mRNA表达水平下调,促凋亡基因Bax和凋亡蛋白caspase一3蛋白及mRNA表达水平上调,Bcl-2／Bax比值增大,与对照组比较,其差异均有统计学意义(P<0.05)。2、与对照组比较,HDL组Cx37、Cx40蛋白和mRNA表达水平均上调,Cx43 mRNA表达水平下调,其差异均具统计学意义(P<0.05)；HDL组明显促进细胞增殖,细胞凋亡率显著下降,细胞存活率增加,与对照组比较,其差异均具有统计学意义(P＜0.05)；线粒体凋亡途径的抑凋亡基因Bcl-2蛋白和mRNA表达水平下调,促凋亡基因Bax和凋亡蛋白caspase-3蛋白及mRNA表达水平上调,Bcl-2／Bax比值增大,与对照组比较,其差异均有统计学意义(P<0.05)。与50μg／ml ox-LDL组比较,HDL+ox-LDL组Cx37、40蛋白和mRNA表达水平上调,Cx43蛋白和mRNA表达水平下调,其差异均具统计学意义(P<0.05)；与50μg／ml ox-LDL组比较,HDL+ox-LDL组细胞增殖并未受抑制,细胞凋亡率下降,细胞存活率增加,其差异均具有统计学意义(P<0.05)；线粒体凋亡途径的抑凋亡基因Bcl-2蛋白和mRNA表达水平上调,促凋亡基因Bax蛋白和mRNA表达水平下调,凋亡蛋白caspase-3 mRNA表达水平下调,Bcl-2／Bax比值增大,与50μg／ml ox-LDL组比较,其差异均有统计学意义(P<0.05)。3、与50μg／ml ox-LDL组比较,octanol+ox-LDL组Cx37、Cx40蛋白和mRNA表达水平上调,Cx43蛋白和mRNA表达水平下调,其差异均具统计学意义(P<0.05); octanol+ox-LDL组细胞增殖抑制率下降,细胞凋亡率下降,细胞存活率增加,与50μg/ml ox-LDL组比较,其差异均具有统计学意义(P<0.05)；线粒体凋亡途径的抑凋亡基因Bcl-2蛋白和mRNA表达水平上调,促凋亡基因Bax、凋亡蛋白caspase-3蛋白和mRNA表达水平下调,Bcl-2/Bax比值增大,与50μg/ml ox-LDL组比较,其差异均有统计学意义(P＜0.05)。结论：1、ox-LDL可以影响人脐静脉内皮细胞Cx蛋白及mRNA表达,且呈剂量依赖性,表明ox-LDL可能通过内皮细胞Cx的重构参与AS的发生和发展。ox-LDL能明显抑制人脐静脉内皮细胞增殖并促其凋亡,且这种作用与浓度相关；其作用机理可能与线粒体凋亡途径Bcl-2\Bax\ Caspase-3有关,ox-LDL可能通过线粒体凋亡途径导致细胞凋亡而参与AS的发生和发展过程。2、HDL能逆转ox-LDL对内皮细胞缝隙连接蛋白和线粒体凋亡途径的影响。HDL和ox-LDL在AS中起相反的作用可能与其通过缝隙连接蛋白对线粒体凋亡途径产生不同的影响有关。3、缝隙连接抑制剂octanol可逆转ox-LDL对人脐静脉内皮细胞缝隙连接蛋白及mRNA的表达水平的影响,阻断缝隙连接后可以抑制ox-LDL对人脐静脉内皮细胞增殖抑制和促凋亡作用,且这种作用可能与线粒体凋亡途径的Bcl-2\Bax\Caspase-3有关。ox-LDL可能通过缝隙连接介导线粒体凋亡途径导致细胞凋亡,促进AS的形成和发展。目的探讨Cx37 C1019T和I1297D基因多态性与动脉粥样硬化性脑梗死发病的相关性。方法通过病例-对照研究设计,采用聚合酶链式反应-限制性片段长度多态性(polymerase chain reaction-restriction fragment length polymorphism, PCR-RFLP)方法检测145例健康对照组人群、245例动脉粥样硬化性脑梗死(atherothrombitic cerebral infarction, ACI)患者Cx37基因C1019T和I1297D位点的单核苷酸多态性。结果1、湖南汉族人群中存在Cx37 C1019T和I1297D基因多态位点,对照组人群中,Cx37 C1019T三种基因型CC、CT和TT频率分别是63.3%、29.0%和9.7%,C和T等位基因频率分别是77.9%和22.1%；Cx37I1297D三种基因型Ⅱ、ID和DD频率分别是47.7%,42.6%和9.7%,I和D等位基因频率分别是69.0%和31.0%。2、Cx37 C1019T在ACI组三种基因型CC、CT和TT频率分别是70.6%,25.7%和3.7%,C和T等位基因频率分别是83.5%和16.5%,与对照组比较,两组基因型频率和等位基因频率无显著性差异(χ2=3.765,P=0.152和χ2=3.692,P=0.055)。ACI组Cx37 I1297D三种基因型Ⅱ、ID和DD频率是45.3%,42.9%和11.8%,与对照组比较,两组基因型频率和等位基因频率无显著性差异(χ2=0.526,P=0.769和χ2=0.458,P=0.499)。结论1、湖南汉族人群中存在Cx37 C1019T和I1297D基因多态位点,且以携带C等位基因和工等位基因为主。2、Cx37 C1019T和I1297D基因多态性可能与湖南汉族人群动脉粥样硬化性脑梗死无关。更多还原

【Abstract】 Stroke is of great concern because of its high incidence, mutilation and mortality rate.60%-80% of stroke is cerebral infarction of which atherosclerosis is the major cause. Thus, thorough understanding of atherosclerosis mechanism can hopefully reduce the incidence of cerebral infarction.During the last decade, tremendous researches have been carried out in elucidating the mechanism of atherosclerosis formation, and in spite of encouraging discoveries of its relation with inflammation and hyperlipidemia and etc, the mechanism still remains unclear.In addition to the endothelium, blood vessel walls are composed of many constituents such as matrix basement, and smooth muscle cells. All these components form a coherent tissue that requires, for proper function, a constant exchange of information via electrical and chemical signals. A key pathway that allows such communication is gap junction (GJ). GJ channels, composed of six connexins to form a channel in the cell membrane, allow the direct exchange of ions small metabolites, and other secondary messengers molecules between cell in contact. Recently, it was found that gap junction was closely related to athereosclerosis, but the mechanism is unclear.On the other hand, a great deal of studies have been focused on the relationship between the Single-nucleotide polymorphism of Connexin 37 (Cx37) and athereosclerosis, coronary heart disease(CHD), Myocardial infarction(MI). It is found that Cx37 C1019T is a Single-gene marker of athereosclerosis, and it is related with CHD and MI. Similar to CHD and MI, cerebral infarction has the common pathologic manifestation of athereosclerosis. However, the relationship between the Single-nucleotide polymorphism of Connexin 37 (Cx37) and athereosclerosis cerebral infarction is unclear.Thus, the influence of oxidized low density lipoprotein (ox-LDL), high-density lipoprotein (HDL) and octanol on the expression of connexin of cultured human umbilical vein endothelial cell were observed and its possible mechanism was studied. Moreover, the relationship between the SNP of Connexin 37 (Cx37) and athereosclerosis cerebral infarction was also studied.Objiective:Our aim was to observe the influence of oxidized low density lipoprotein(ox-LDL) on the expression of connexin and the apotosis of cultured Human umbilical vein endothelial cell (HUVE-12), as well as to investigate the influence and the mechanism of HDL and octanol on the expression of connexin and apotosis of cultured endothelial cells induced by ox-LDL.Methods:HUVE-12 cells were divided into seven groups and were exposed to different conditions. The groups included:①control group: normal DMEM culture medium;②-④ox-LDL group:HUVE-12 were incubated with 25μg/ml ox-LDL,50μg/ml ox-LDL and 100μg/ml ox-LDL for 24 hours;⑤HDL group:HUVE-12 was incubated with 100μg/ml HDL for 24 hours;⑥HDL and ox-LDL group:HUVE-12 were coincubated with 100μg/ml HDL and 50μg/ml ox-LDL for 24 hours;⑦octanol and ox-LDL group:HUVE-12 was coincubated with 1mm octanol and 50μg/ml ox-LDL for 24 hours. MTT method was carried out to evaluate the proliferation of HUVE-12 while viability of HUVE-12 was detected by Trypan blue staining, cell apotosis was examined through flow-cytometry. The expressions of Cx37,40,43, Bcl-2, Bax and Caspase-3 mRNA were examined by real time RT-PCR, also the proteins expression was examined by immunocy-tochemistry and western blot.Result:1. Expressions of proteins and mRNA of Cx37 and Cx 40 were decreasing when HUVE-12 incubated with 25μg/ml,50μg/ml and 100μg/ml ox-LDL for 24h. By contrast, expressions of proteins and mRNA of Cx 43 were significantly elevated comparing with the control group (P<0.05). The results showed that after HUVE-12 were exposed to various concentration of ox-LDL for 24h, cell proliferation was downregulated and its viability decreased significantly in a concentration-dependent manner while cell apotosis was increased (P<0.05). Expressions of proteins and mRNA of Bcl-2 were decreased, while expressions of proteins and mRNA of Bax and caspase-3 were increasing. The specific values for Bcl-2/Bax in groups with different ox-LDL concentrations were significantly elevated (P<0.05).2. Comparing with the 50μg/ml ox-LDL group, expressions of proteins and mRNA of Cx37 and Cx 40 in HDL group were upregulated, when the mRNA expression of Cx43 was downregulated (P＜0.05). Cell proliferation and viability in HDL group were enhanced significantly while cell apotosis was ameliorated (P＜0.05). Expressions of proteins and mRNA of Bcl-2 were upregulated, while the expressions of proteins and mRNA of bax and caspase-3 were downregulated. The specific values for Bcl-2/Bax was elevated (P<0.05). Expressions of proteins and mRNA of Cx37 and Cx 40 in HDL+ox LDL group were upregulated, when expressions of protein and mRNA of Cx43 were downregulated (P<0.05). Cell proliferation and viability in HDL+ ox-LDL group were enhanced significantly while cell apotosis was decreased (P＜0.05). Expressions of proteins and mRNA of Bcl-2 were upregulated, while expressions of proteins and mRNA of bax were downregulated, and mRNA expression of caspase-3 was upregulated. The specific values for Bcl-2/Bax was elevated (P＜0.05).3. Comparing with the 50μg/ml ox-LDL group, expressions of proteins and mRNA of Cx37 and Cx 40 in octanol+ox-LDL group were upregulated, when expressions of proteins and mRNA of Cx43 were downregualted (P＜0.05). What’s more, Cell proliferation and viability in octanol+ox-LDL group were enhanced significantly while cell apotosis was decreased (P<0.05). Expressions of proteins and mRNA of Bcl-2 were increased, while expressions of proteins and mRNA of bax and caspase-3 were decreased. The specific values for Bcl-2/Bax was elevated significantly (P＜0.05).Conclusion:1. ox-LDL could induce the remodeling of connexin in HUVE-12 in virto, and the effect was in a concentration-dependent manner. It was suggested that ox-LDL could lead to AS by the remodeling of connexin. HUVE-12 cell proliferation and viability were significantly downregulated in a concentration-dependent manner by ox-LDL while cell apotosis was enhanced. The effect of ox-LDL on cell apotosis of HUVE-12 could be related to mitochondrial apoptosis pathway which could be a mechanism of AS causing by ox-LDL.2. HDL could prevent the remodeling of connexin in HUVE-12 and decrease cell apotosis of HUVE-12 induced by ox-LDL. It indicated that HDL could play a role in anti-AS by this pathway.3. Similarly with the effect of HDL, octanol could prevent the remodeling of connexin and decrease cell apotosis in HUVE-12 induced by ox-LDL. The effect of octanol on cell apotosis caused by ox-LDL could also be related to mitochondrial apoptosis pathway. ox-LDL could affect connexins and gap junction in endothelial cells and it could result in the chain of mitochondrial apoptosis pathway which lead to cell apotosis.Objective:Our aim was to investigate the relationship between connexin 37 gene polymorphisms and atherothrombotic cerebral infarction.Methods:A case-control study was carried out in which connexin 37 C1019T and I1297D gene polymorphisms in 245 paitents with atherothrombotic cerebral infarction (ACI group) and 145healthy subjects (control group) were analyzed with polymerase chain reaction (PCR)-restriction fragment length polymorphism(RFLP).Result:1. The results showed that there were Cx37 C1019T and I1297D single-nucleotide polymorphism (SNP) in han population of Hunan province. In control group Cx37 C1019T CC, CT and TT genotype frequencies were 63.3%、29.0% and 9.7%, while C and T allele frequencies were 77.9% and 22.1%. Cx37 I1297D II, ID and DD genotype frequencies were 47.7%,42.6% and 9.7%, while I and D allele frequencies were 69.0% and 31.0%.2. In ACI group Cx37 C1019T CC, CT and TT genotype frequencies were 70.6%,25.7% and 3.7%,, while C and T allele frequencies were 83.5% and 16.5%. But no differences were detected of genotypes and allele frequencies among patients with ACI group and control group (x 2=3.765, P=0.152 and x 2=3.692, P=0.055). Cx37 I1297DⅡ, ID and ID genotype frequencies were 45.3%,42.9% and 11.8%, while I and D allele frequencies were 66.7%and 33.3%. But no differences were detected of those genotypes and allele frequencies among patients with ACI group and control group (x 2=0.526,P=0.769 and x 2=0.458,P=0.499).Conclusion1. There were Cx37 C1019T and I1297D single-nucleotide polymorphism (SNP) in han population of Hunan Province. The Cx37 C1019T C allele and Cx37 I1297D I allele were dominant respectively in both groups.2.Cx37gene C1019T和I1297D polymorphism were not associated with an increased risk of CI in Han population of Hunan Province, China, and might not be considered as an independent predictor of atherothrombotic cerebral infarction.更多还原