p22phox在高糖所致的内皮细胞凋亡中的作用及机制

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1、分类号密级UDC编号硕士学位论文p22phox在高糖所致的内皮细胞凋亡中的作用及机制研究生姓名：盛顺良指导教师姓名、职称：王佐教授学科、专业名称：病理学与病理生理学研究方向：动脉粥样硬化发病机制研究2013年4月1南华大学学位论文原创性声明本人声明，所呈交的学位论文是本人在导师指导下进行的研究工作及取得的研究成果。尽我所知，除了论文中特别加以标注和致谢的地方外，论文中不包含其他人已经发表或撰写过的研究成果，也不包含为获得南华大学或其他单位的学位或证书而使用过的材料。与我共同工作的同志对本研究所作

2、的贡献均已在论文中作了明确的说明。本人完全意识到本声明的法律结果由本人承担。作者签名：年月日南华大学学位论文版权使用授权书本人同意XX大学有关保留、使用学位论文的规定，即：学校有权保留学位论文，允许学位论文被查阅和借阅；学校可以公布学位论文的全部或部分内容，可以编入有关数据库进行检索，可以采用复印、缩印或其它手段保留学位论文；学校可根据国家或湖南省有关部门规定送交学位论文。对于涉密的学位论文，解密后适用该授权。作者签名：导师签名：年月日年月日2目录一、中文摘要„„„„„„„„„„„„„„„„„„

3、„„4二、英文摘要„„„„„„„„„„„„„„„„„„„„„6三、正文„„„„„„„„„„„„„„„„„„„„„„„8前言„„„„„„„„„„„„„„„„„„„„„„„„8材料与方法„„„„„„„„„„„„„„„„„„„„10实验结果„„„„„„„„„„„„„„„„„„„„„16讨论„„„„„„„„„„„„„„„„„„„„„„„23结论„„„„„„„„„„„„„„„„„„„„„„„26四、参考文献„„„„„„„„„„„„„„„„„„„„„27五、附录„„„„„„„„„„„„„„„„„„„„„„„

4、31六、文献综述„„„„„„„„„„„„„„„„„„„„„32七、发表论文„„„„„„„„„„„„„„„„„„„„„43八、致谢„„„„„„„„„„„„„„„„„„„„„„„443p22phox在高糖所致的内皮细胞凋亡中的作用及机制研究生：盛顺良导师：王佐教授中文摘要目的探讨p22phox基因沉默在高浓度葡萄糖诱导的内皮细胞活性氧生成和细胞凋亡中的作用及其机理。方法用Ⅱ型胶原酶消化分离培养人脐静脉内皮细胞（HUVECs），在倒置显微镜下观察其形态。免疫组化法检测HUVECs内第Ⅷ因子相关抗原的表

5、达。将原代培养的人脐静脉内皮细胞分为对照组、高糖组、高糖+siRNA转染组和siRNA转染组。采用Western-blot检测p22phox和caspase-3蛋白的表达，流式细胞仪检测细胞内活性氧水平及细胞凋亡率，Hochest33342染色观察细胞的胞核形态，分光光度计检测caspase-3酶活性。结果分离得到的HUVECs均表达第Ⅷ因子相关抗原；siRNA能有效抑制p22phox表达；高葡萄糖能诱导p22phox表达；与对照组相比，高糖组细胞p22phox蛋白水平、活性氧水平、细胞凋亡率、

6、caspase-3活性和蛋白水平均明显增高（P<0.05）；高糖+siRNA转染组的细胞p22phox蛋白水平、活性氧水平、细胞凋亡率、caspase-3活性和蛋白水平均明显低于高糖组（P<0.05）。结论1.转染siRNA-p22phox能抑制内皮细胞p22phox表达；2.抑制p22phox表达能降低高糖所致的内皮细胞活性氧生成和细胞凋亡。关键词动脉粥样硬化，人脐静脉内皮细胞，p22phox，葡萄糖，活性氧4本课题由以下基金项目资助本课题受湖南自然科学基金(№:30470720)；中国博士后

7、基金(№:2005037157)；湖南省自然科学基金(№:06jj5058)；湖南省教育厅课题(07C617)资助，在此予以特别致谢。5TheEffectsofp22phoxonEndothelialCellApoptosisInducedbyHigh-glucoseandItsMechanismsObjectives:TostudytheeffectsofNAD(P)Hoxidasesubunitsp22phoxonendothelialROSinductionandcellapoptosis

8、inducedbyhigh-glucose.Methods：MophologyofHUVECswasobservedbyinvertedmicroscope.ExpressionoffactorⅧrelatedantigeninHUVECswasinvestigatedbyimmunohistochemistry.HUVECsweredividedintofourgroups:controlgroup,highglucosegroup,siRNAgroupandhighglucose+siRNA