摘要
目的观察cubilinsiRNA真核表达载体对人近端肾小管上皮细胞(human renal proximal tubular epithelial cell line,HKC)cubilin基因的特异性抑制作用。方法针对人cubilin基因设计并构建siRNA真核表达载体pSUPER EGFP-C1和pSUPER EGFP-C2,转染HKC,经荧光定量PCR(FQ-PCR)检测cubilin mRNA的表达、不同浓度的人血清白蛋白(HSA)刺激正常HKC及转染重组质粒的HKC24h后cubilin mRNA的表达,并通过激光共聚焦扫描荧光显微镜观察HKC对白蛋白摄取情况的变化。结果FQ-PCR显示HSA刺激正常HKC后cubilin mRNA表达具有剂量依赖性。与正常对照组相比,两个重组质粒均可部分抑制HKC cubilin表达,抑制效率分别为66%和37%,HSA刺激转染pSUPER EGFP-C1、pSU-PER EGFP-C2的HKC后cubiin mRNA水平较正常对照组比较分别下降50%、34%,转染了重组质粒的HKC对白蛋白的摄取明显减少。结论成功地构建了针对人cubilin基因的RNA干扰真核表达载体,表达型siRNA可抑制肾小管上皮细胞cubilin基因的表达及对白蛋白的摄取。
Objective To construct eukaryotic expression vector of siRNA for human cubilin gene and detect its specific interference in human renal proximal tubular epithelial cell line （HKC）. Methods Two eukaryotic expression vectors of siRNA were constructed for human cubilin gene. HKCs were stably transfected with the pSUPER EGFP vector and the two recombinant vectors. Normal HKCs and transfected HKCs were stimulated by human serum albumin （HSA）. Then cubilin mRNA expression of HKCs were detected by fluores- cence-based quantitative RT-PCR （FQ-PCR）. Endocytosis of TRITC labeled bovine serum albumin （ TRITC- BSA） by HKCs was detected by laser scanning confocal fluorescence microscope. Results DNA sequencing showed that the target segments were cloned into pSUPER EGFP vector respectively. The results of FQ-PCR indicated there was dose-dependent cubilin mRNA expression in HKCs stimulated with HSA and both vectors could inhibit expression of cubilin gene. The expression of cubilin obviously decreased by 66% and 37% respectively in the transfected HKCs with the two recombinant vectors. TRITC-BSA uptake was obviously lower inHKCs transfected with two recombinant vectors. Conclusion The eukaryotic expression vector of siRNA for human cubilin gene was successfully constructed. The expression of cubilin gene and albumin uptake were inhibited effectively by the expressed siRNA.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2007年第6期471-474,共4页
Journal of Third Military Medical University
基金
国家自然科学基金(30370534)~~
