【作者】 杜建玲；

【导师】 安利佳；

【作者基本信息】 大连理工大学 ， 生物化工， 2008， 博士

【摘要】 内皮细胞功能障碍（Endothelial cell dysfunction,ECD）在糖尿病（Diabetesmellitus,DM）血管并发症的发生和发展中起着重要作用,探求有效的内皮保护策略已受到学术界普遍关注。本研究旨在（1）探讨中国人脂肪组织中硒蛋白S（Selenoprotein S,SelS）基因的表达及其与血清淀粉样蛋白（Serum Amyloid A,SAA）和胰岛素抵抗（Insulin resistance,IR）的相关性,为2型糖尿病（Type 2Diabetes Mellitus,T2DM）及其大血管并发症发病机制和防治研究提供新的方向;（2）应用分子生物学技术分离SelS基因,构建SelS真核表达载体——pLNCX2-SelS,并将其转染至人脐静脉内皮细胞(HUVECs,ECV₃₀₄细胞),通过DNA序列分析、mRNA及蛋白水平检测进行鉴定,获得高表达SelS的细胞株,为研究SelS的功能提供可靠的技术支持;（3）建立外源性过氧化氢（HydrogenPeroxide,H₂O₂）刺激诱导的细胞损伤模型,探讨高表达SelS对ECV₃₀₄细胞的抗氧化保护作用及机制,为T2DM血管并发症的防治建立新的策略和方法。应用RT-PCR技术,检测12例非糖尿病患者及10例T2DM患者大网膜脂肪组织中SelS基因的表达;计算胰岛素抵抗指数（Homa-IR）,ELISA测定血清SAA的水平。结果显示,T2DM患者大网膜脂肪组织中SelS表达水平、Homa-IR及血清SAA水平均高于非糖尿病对照组;并且,在两组的大网膜组织中SelS表达水平与Homa-IR和血清SAA水平均呈正相关。提示中国T2DM患者大网膜脂肪组织表达SelS与IR有关,结合文献报告推测SelS可能是作为SAA的受体在T2DM和大血管并发症的发生发展中发挥作用。取皮下脂肪组织提取总RNA,RT-PCR扩增SelS基因,回收并命名为SelSInsert DNA1。SelS Insert DNA1被克隆至克隆载体pMD18-T,经DNA序列分析鉴定结果无误;将pMD18-SelS质粒进行大量扩增酶切,切胶回收目的基因片段,命名为SelS Insert DNA2。SelS Insert DNA2亚克隆至真核表达载体pLNCX2,经PCR反应以及DNA序列分析鉴定结果,证实真核表达载体pLNCX2-SelS构建正确。将pLNCX2-SelS转染至ECV₃₀₄细胞,RT-PCR和Western blot检测进一步证实重组SelS真核表达载体pLNCX2-SelS成功表达;同时发现,SelS基因在pLNCX2-SelS转染组的表达水平是ECV₃₀₄细胞内源性表达水平的1.76倍（mRNA表达）和1.56倍（蛋白表达）。应用不同浓度的H₂O₂损伤ECV₃₀₄细胞后,高表达SelS组能显著增加细胞活性、减少H₂O₂诱导ECV₃₀₄细胞丙二醛（MDA）的产生、增加超氧化物歧化酶（SOD）的活性、抑制H₂O₂诱导的窖蛋白-1（Caveolin-1）上调;提示SelS可通过抗氧化作用保护内皮细胞免于H₂O₂介导的细胞损伤,其保护机制可能与SelS抑制Caveolin-1的上调有关。上述研究表明,内脏脂肪组织SelS高表达与T2DM患者的IR有关,并可能是作为SAA的受体在T2DM和大血管并发症的发生发展中发挥一定的作用;将从人脂肪组织中克隆出的SelS基因片段插入到逆转录病毒载体pLNCX2中,成功构建pLNCX2-SelS重组真核表达载体,并获得SelS高表达细胞株;ECV₃₀₄细胞中有内源性SelS表达,高表达SelS可以明显减弱H₂O₂诱导的ECV₃₀₄细胞的损伤;高表达SelS抗氧化保护ECV₃₀₄细胞的作用与抑制Caveolin-1上调有关。更多还原

【Abstract】 Endothelial cell dysfunction（ECD） plays an important role in development of diabetic vascular complications.More and more researchers are interested in how to pretect endothelial cell effectively.The purposes of this study are shown as the followings:（1） To study on correlation between SelS mRNA expression in Chinese adipose tissue,Insulin resistance（IR） and Serum Amyloid A（SAA）,which provides a new direction for preventing the development of type 2 diabetes mellitus（T2DM） and atherosclerosis.（2） To construct the Eukaryotic expression vector of Selenoprotein S（SelS） and transfect into human umbilical vein endothelium cells (HUVECs,ECV₃₀₄ Cells) by using molecular biological technology and identify the DNA sequence analysis,mRNA and protein level of SelS,which provides a reliable technique support for SelS study.（3） To establish the cellular damage model induce by exogenous hydrogen peroxide（H₂O₂） and study the over-expression of SelS in the protective function and mechanism of ECV₃₀₄ cell,which provides a new strategy for preventing the development of T2DM and atherosclerosis.The following studies of human omental adipose tissues from ten cases of type 2 diabetic patients and twelve cases of non-diabetic individuals were carried on to examine the expression level of Tanis/SelS mRNA by semi-quantitative PCR, calculate Homa-IR by standard formula and detect SAA level by ELISA.Tanis/SelS mRNA expression,Homa-IR and serum SAA were higher in T2DM than in control group.Tanis/SelS mRNA level was positively correlated with Homa-IR and SAA level in two groups.SelS protein might involve in insulin resistance in Chinese with T2DM,possibly acting as the SAA receptor,thus play an important role in the development of T2DM and atherosclerosis.After total RNA was isolated from human subcutaneous adipose tissue,SelS gene segment,which was called SelS Insert DNA1,was amplified by RT-PCR and then cloned into the vector of pMD18-T.Recombinant plasmid of pMD18-SelS was confirmed by DNA sequence analysis.SelS Insert DNA2,which was purified successively by plasmid amplified,restrictive digestion and gel extraction,was subcloned into Eukaryotic expression vector of pLNCX2.After identified by PCR and DNA sequence analysis,the recombinant plasmid of pLNCX2-SelS was transfected into ECV₃₀₄ cell and SelS were expressed successfully both in mRNA and protein levels.Moreover,the gene expression level of SelS was 1.76 fold in transfection group compared to endogenous gene expression and the protein expression level was 1.56 fold.After ECV₃₀₄ cells were damaged by different concentration of H₂O₂,both of the activity of cellular proliferation and SOD were increased in the group of over-expressed SelS gene compared with control one.Furthermore,elevated SelS gene expression in ECV₃₀₄ cells could attenuate the production of MDA and inhibit the up-regulation of Caveolin-1 induced by H₂O₂.It is suggested that SelS might protect endothelial cells from H₂O₂-induced impairment through anti-oxidative pathway and this protective mechanism might related to the depressing the elevation of Caveolin-1 by SelS.Therefore,up-regulated SelS in adipose might involve in insulin resistance, possibly acting as the SAA receptor,thus play an important role in the development of T2DM and atherosclerosis.SelS gene fragment isolated from human subcutaneous adipose was inserted into retrovirus vector of pLNCX2 and recombinant Eukaryotic expression vector of pLNCX2-SelS was successfully constructed.Elevated SelS, which was also expressed endogenously in ECV₃₀₄ cells,could attenuate the damage induced by H₂O₂ and play a role in ECV₃₀₄ cellular anti-oxidative protection through preventing the elevation of Caveolin-1.更多还原