9型重组腺相关病毒介导抗核转录因子-κB核酶基因体外转染大鼠心肌细胞及对核转录因子-κB活性的影响  

Transfection of rats H9C2 cells with recombinant adeno-associated virus Serotype 9 mediated AntiNF-κB ribozyme in vitro and effects on nuclear factor-κB activity

作　　者：高霞[1] 马依彤[1] 杨毅宁[1] 向阳[1] 陈邦党[1] 刘芬[1] 

机构地区：[1]新疆医科大学第一附属医院心脏中心,乌鲁木齐830054

出　　处：《中华实验外科杂志》2010年第9期1227-1229,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30760263）;新疆自治区卫生厅青年科技人才专项基金资助项目（2007Y23）;新疆维吾尔自治区重点实验室资助项目（XJDX0903）

摘　　要：目的 观察9型重组腺相关病毒（rAAV9）介导抗核转录因子-κB（NF-κB）核酶基因（rAAV9-ECFP-R65）对大鼠心肌H9C2细胞的转染及对NF-κB活性的影响.方法 rAAV9-EGFP-R65按转染复数（MOI）1×10^6v.g./cell转染H9C2细胞,在倒置荧光显微镜下观察增强型绿色荧光蛋白（EGFP）阳性表达,采用流式细胞仪检测转染效率.Alamar Blue法检测rAAV9-EGFP-R65对H9C2细胞增殖影响.肿瘤坏死因子-α（TNF-α）、rAAV9-EGFP-R65及PDTC处理H9C2细胞,凝胶迁移滞后实验（EMSA）检测NF-κB活性.结果 转染后第1天EGFP开始表达,表达强度随着时间延长而逐渐增强,第5天达到高峰,此时流式细胞仪检测转染率为（32.27±3.19）%.Alamar Blue法检测表明转染组细胞增殖末受影响.常态下H9C2细胞NF-κB有一定活性,TNF-α刺激后NF-κB活性明显增高,而rAAV9-EGFP-R65、PDTC抑制NF-κB活性.结论 rAAV9-ECFP-R65能够有效地转染H9C2细胞,对细胞增殖无明显抑制,并能显著抑制H9C2细胞NF-κB活性.Objective To evaluate the transfection efficiency using recombinant adeno-associated virus serotype 9 （rAAV9） mediated anti-nuclear factor-κB （NF） -κB ribozyme and enhanced green fluorescent protein （rAAV9-EGFP-R65） to rats H9C2 cells and the effect on NF-κB activity. Methods rAAV9EGFP-R65 was transfected into H9C2 ceils at multiplicities of infection （ MOI = 1 x 10^6 v. g./cell）. EGFP expression in the cells was observed under an inverted fluorescence microscope, and the percentage of EGFP positive cells was determined by flow cytometry. Alamar Blue assay was used to assess the proliferation of the transfected cells. H9C2 ceils were treated with tumor necrosis factor （TNF）-α, rAAV9-EGFP-R65 and PDTC. The DNA binding activity of NF-KF-κB was examined by electrophoretic mobility shift assay （EMSA）. Results The cells began to exhibit EGFP expression one day after transfection. The fluorescence intensity was increased with the time of transfection. EGFP expression reached the maximum on the day 5, at the point of which the transduction efficiency was （32.27 ＋ 3.19）%. Alamar Blue assay did not reveal significant difference in the absorbance between the transfected cells and the control cells. TNF-α could activate NF-κB, and rAAV9-EGFP-R65 and PDCT could efficiently decrease NF-κB activation in rats H9C2 cells. Conclusion rAAV9-EGFP-R65 can be stably and efficiently expressed in H9C2 cells without causing cell growth inhibition, rAAVg-EGFP-R65 can availably inhibit NF-κB activation in rats H9C2 cells in vitro.

关 键 词：重组腺相关病毒 核酶 心肌细胞 转染 核转录因子-κB 

分 类 号：R54[医药卫生—心血管疾病]