糖尿病线粒体ND1基因3316G→A突变的功能研究  

Study on mitochondrial function of ND1 gene with 3316 G→A mutation in human diabetes

作　　者：邓冠华[1] 周新[1] 庞志宇[1] 刘松梅[1] 谢焱[1] 

机构地区：[1]武汉大学中南医院临床基因诊断中心,430071

出　　处：《中华医学杂志》2009年第40期2822-2826,共5页National Medical Journal of China

基　　金：基金项目：国家自然科学基金（30672012）

摘　　要：目的探讨线粒体ND1基因3316G—A突变对线粒体生物学功能的影响及其在糖尿病发病中的作用。方法利用真核表达载体pcDNA3．1B和大肠杆菌DH5α构建野生型和线粒体DNA的ND1基因3316G—A突变型重组子pcDNA3．1B—ND1；设计特异性siRNA序列干扰Hela细胞内源性线粒体DNA表达，使其内源性ND1基因沉默；经脂质体转入野生型和突变型重组子pcDNA3．1B—NDl，使其在Hela细胞内表达。通过RT—PCR、十二烷基硫酸钠一聚丙烯酰胺凝胶电泳和荧光显微镜，从mRNA水平→蛋白质水平→线粒体膜电势位检测干扰效果，以筛选有效干扰siRNA序列。结果线粒体ND11和线粒体ND12siRNA序列均具有一定的干扰效果，后者效果明显优于前者；转入3316G→A突变型重组子pcDNA3．1B—ND1的Hela细胞表达的线粒体蛋白低于野生型。结论线粒体DNA的ND1基因正常表达对维持正常的呼吸链功能和细胞增殖至关重要，携有3316G→A突变基因的糖尿病的发病与线粒体蛋白表达下降有关。Objective To explore the effects of ND1 gene with 3316 G→A mutation upon mitochondrial function and elucidate its role in the development of human diabetes. Methods The eukaryotic expression vector pcDNA3.1B and E. coli DH5α were used to construct the reconbinant plasmid （pcDNA3.1B-ND1） of wild-type and 3316 G→A mutant type ND1 gene. And the recombinant plasmids were analyzed by restriction enzyme digestion and DNA sequencing. Two siRNAs （mtND11 and mtND12） specific for human mtNDA ND1 gene were designed, synthesized and then transfected into Hela cells for silencing endogenous mtDNA ND1 gene. The gene-silencing effects were analyzed by RT-PCR, SDS-PAGE and MitoCaptureTM mitochondrial apoptosis detection kit. Later the two types recombinant plasmids were transfected into Hela cells in which endogenous mtDNA ND1 gene was silenced. After 48 h culture, the Hela cells were collected for determination of mitochondrial proteins by SDS-PAGE. Results Both mtND11 and mtND12 could decrease mtDNA ND1 expression and mtND11 caused a smaller decrease. The expression of mitochondrial protein in 3316 G→ A mutant type recombinant decreased. Conclusion The normal expression of mitochondrial NDI gene maintain the function of mitochondrial respiratory chain and cell proliferation. The 3316 G→A mutation in mitochondrial NDlgene might be related to the down-regulated expression of mitochondrial protein and the diabetes mellitus pathogenesis.

关 键 词：糖尿病 DNA 线粒体 基因 

分 类 号：R686[医药卫生—骨科学]