CAJ | 학술논문

目的探讨线粒体ND1基因3316 G→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3316 G→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-ND1,使其在Hela细胞内表达.通过RT-PCR、十二烷基硫酸钠-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ND12 siRNA序列均具有一定的干扰效果,后者效果明显优于前者;转入3316 G→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3316 G→A突变基因的糖尿病的发病与线粒体蛋白表达下降有关.
목적 탐토선립체ND1기인3316 G→A돌변대선립체생물학공능적영향급기재당뇨병발병중적작용.방법 이용진핵표체재체pcDNA3.1B화대장간균DH5α구건야생형화선립체DNA적ND1기인3316 G→A돌변형중조자pcDNA3.1B-ND1;설계특이성siRNA서렬간우Hela세포내원성선립체DNA표체,사기내원성ND1기인침묵;경지질체전입야생형화돌변형중조자pcDNA3.1B-ND1,사기재Hela세포내표체.통과RT-PCR、십이완기류산납-취병희선알응효전영화형광현미경,종mRNA수평→단백질수평→선립체막전세위검측간우효과,이사선유효간우siRNA서렬.결과 선립체ND11화선립체ND12 siRNA서렬균구유일정적간우효과,후자효과명현우우전자;전입3316 G→A돌변형중조자pcDNA3.1B-ND1적Hela세포표체적선립체단백저우야생형.결론 선립체DNA적ND1기인정상표체대유지정상적호흡련공능화세포증식지관중요,휴유3316 G→A돌변기인적당뇨병적발병여선립체단백표체하강유관.
Objective To explore the effects of ND1 gent 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 Heia 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 ceils 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 mitoehondrial ND1 gene maintain the function of mitochondrial respiratory chain and cell proliferation.The 3316 G→A mutation in mitoehondrial Ndlgene might be related to the down-regulated expression of mitoehondrial protein and the diabetes mellitus pathogenesis.