敲低QDPR基因慢病毒构建及其干涉NRK-52E细胞效果研究  

QDPR RNA INTERFERENCE LENTIVIRAL VECTORS CONSTRUCTION AND ITS INTERFERENCE EFFECT ON NRK-52E CELL

作　　者：蒲志杰 张景义[1] 孙耀东[2] 杨振邦[2] 杨向君[3] 何海兰[2] 刘业强[1] 孟令宇[1] 沈宏[4] 杨方[3] 李治国[3] 

机构地区：[1]河北联合大学附属开滦总医院内分泌科,河北省唐山市063000 [2]河北联合大学临床医学及实验技术系 [3]河北联合大学医学实验中心 [4]河北联合大学信息中心

出　　处：《中国煤炭工业医学杂志》2014年第12期1996-1999,共4页Chinese Journal of Coal Industry Medicine

基　　金：国家自然科学基金青年基金(编号:81100518);唐山市科学研究与发展计划项目(编号:111302112b);河北省高等学校技术研究青年基金项目(编号:QN2014013)

摘　　要：目的构建醌型二氢生物碟呤还原酶(quinonoid dihydropteridine reductase,QDPR)敲低慢病毒模型,检测其对肾小管上皮NRK-52E细胞QDPR基因的敲低情况。方法设计合成QDPR的干涉shRNA序列,并重组入GP2慢病毒载体质粒,测序检验重组质粒是否转入该序列。三质粒共转染获得干涉慢病毒,将其感染NRK-52E细胞,新霉素筛选抗性细胞,用实时定量PCR、western blot和QDPR酶活性测量检测干涉效果。结果重组入GP2的序列与设计序列一致,病毒转染NRK-52E细胞使其获得了新霉素抗性,QDPR基因表达、蛋白含量及酶活性显著降低。结论本研究合成的QDPR基因敲低慢病毒能使NRK-52E细胞QDPR基因的mRNA转录、蛋白浓度及活性降低,为后续QDPR基因在糖尿病肾病中的作用研究提供了模型。Objective To construct quinonoid dihydropteridine reductase（QDPR）interference lentiviral vectors and examine its interference effect on NRK-52 E cell.Methods QDPR interference shRNA was designed and synthesized.The sequence was recombined to GP2 plasmids and identified by sequencing.Three plasmids were co-transfected to obtain interference lentivirus,and infect NRK-52 E cells.Neomycin resistant was test on virus infected NRK-52 E cells.Real-time quantitative PCR,western blot and enzyme activity measurements were used to screen QDPR interference effect.Results The sequences recombinant into GP2 is consistent with the design sequence.NRK-52 E cells transfected with the virus obtained the ability of neomycin resistance.And they significantly reduce QDPR gene expression,protein concentration and activity.Conclusion This study constructed QDPR gene knockdown lentivirus.Infected with the virus,NRK-52 E cells reduce QDPR mRNA expression,protein concentrating and activity.This will provides a useful model for subsequent QDPR gene functional study in diabetic nephropathy.

关 键 词：醌型二氢生物碟呤还原酶 慢病毒 糖尿病肾病 肾小管上皮细胞 

分 类 号：R587.1[医药卫生—内分泌]