共转染小干扰RNA质粒联合抑制HeLa细胞葡萄糖-6-磷酸脱氢酶和转酮醇酶样基因-1表达  

Combined inhibitory effect of co-transfected siRNA plasmids in Hela cells on the expressions of glucose-6-phosphate dehydrogenase and transketolase like-1

作　　者：张德太[1] 杨文[1] 涂启明[1] 张科[1] 胡丽华[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院检验科,武汉医学博士430022

出　　处：《医学研究生学报》2013年第11期1156-1160,共5页Journal of Medical Postgraduates

摘　　要：目的葡萄糖-6-磷酸脱氢酶(glucose-6-phosphate dehydrogenase,G6PD)及转酮醇酶样基因-1(transketolase like-1,TKTL-1)是肿瘤细胞磷酸戊糖代谢途径(pentose phosphate pathway,PPP)的2个重要关键调节酶。拟通过共转染小干扰RNA(small interfering RNA,siRNA)表达载体同时沉默人宫颈癌Hela G6PD及TKTL-1后,观察其对G6PD和TKTL1的联合抑制作用,旨在探讨共转染siRNA表达载体联合抑制PPP的效果及可行性。方法分别构建靶向G6PD和TKTL1基因siRNA干扰质粒载体,通过酶切和测序鉴定siRNA干扰质粒载体构建成功后,单独或共转染HeLa细胞株,RT-PCR检测G6PD、TKTL1 mRNA表达并结合G6PD、转酮醇酶(transketolase,TKT)活性测定判断并比较共转染联合抑制Hela细胞G6PD、TKTL1表达的效果。结果共转染靶向G6PD和TKTL1基因的siRNA干扰载体能显著下调G6PD和TKTL1基因表达水平[(0.96±0.05)vs(0.47±0.04),(0.98±0.05)vs(0.41±0.04),P<0.01]。同时,2个酶的活性也显著下降(99.2%vs34.5%和99.8%vs 40.1%,P<0.01)。共转染抑制G6PD基因mRNA表达较单独转染靶向G6PD基因的siRNA干扰载体的效果有显著下降[(0.47±0.04)vs(0.31±0.04),P<0.01)]。结论共转染siRNA干扰载体对人宫颈癌HeLa细胞PPP代谢通路进行联合抑制是一种有效的实验方案。Objective Glucose-6-phosphate dehydrogenase （G6PD） and transketolase like-1 （TKTL1） are key regulatory en- zymes of the pentose phosphate pathway （PPP） in tumors. This study observed the metabolic changes and biologic characteristics in tumor cells by silencing C,6PD and TKTL1 genes, evaluated the combined inhibitory effect of co-transfected siRNA plasmids in Hela cells on G6PD and TKTL1 expressions, and investigated the effect and practicality of the combined suppression of PPP using eo-transfected siRNA plasmids. Methods Specific siRNA expressing vectors targeting G6PD and TKTL1 genes were separately constructed and the recombi- nant plasmids were verified by restriction endonuclease and DNA sequencing. Then the recombinant plasmids were transfected into Hela cell line separately or jointly; their combined inhibitory effect on G6PD and TKTL1 expressions were evaluated by comparing their activi-ties and mRNA expressions using RT-PCR. Results After joint transfection of the two types of recombinant plasmids, the G6PD and TKTL1 mRNA expressions were decreased significantly （0.96 ± 0.05 vs 0.47 ±0.04, 0.98±0.05 vs 0.41±0.04, both P〈0.01）. Simul-taneously, the activities of G6PD and TKT were also statistically decreased （99.2% vs 34.5%, 99.8% vs 40. 1%, both P 〈0.01 ）. Compared with the single G6PD siRNA expression vector transfection group, G6PD mRNA expressions in the co-transfected group were remarkably reduced （0.47 ± 0.04 vs 0.31 ± 0.04, P 〈 0.01 ）. Conelusion Co-transfection of siRNA plasmids in Hela cells was an ef- fective experimental program for the combined suppression of PPP.

关 键 词：小干扰RNA 转酮醇酶样基因-1 葡萄糖-6-磷酸脱氢酶 HELA细胞 

分 类 号：Q786[生物学—分子生物学]