摘要
目的:利用短发夹RNA干扰载体介导的RNA干扰技术沉默成年大鼠心脏成纤维细胞中黏附斑激酶,观察细胞增殖和凋亡的变化。方法:实验于2004-12/2005-12在解放军第三军医大学新桥医院中心实验室完成。实验选用10～12周龄清洁级的雄性Wistar大鼠24只,以转染靶向黏附斑激酶的重组RNA干扰质粒的心脏成纤维细胞为干预组,选择未转染细胞和转染空质粒、非特异siRNA的细胞为不同对照组,共4组,各6只。消化法培养大鼠心脏成纤维细胞,设计、构建和鉴定黏附斑激酶RNA干扰载体,westernblot法测量黏附斑激酶的沉默效率,四唑盐比色法检测细胞增殖,DNA缺口末端标记技术检测细胞凋亡,流式细胞仪测定B细胞淋巴瘤2和bax的变化。结果:Wistar大鼠24只,全部进入结果分析。①酶切鉴定和测序鉴定表明黏附斑激酶RNA干扰载体构建成功,转染细胞后,Westernblot结果表明第二个重组黏附斑激酶RNA干扰载体的RNA沉默效率最高,为72.1%,其余分别为66.2%和55.3%。②Pavu6+27-黏附斑激酶2转染组A490值明显低于未转染细胞和转染空质粒、非特异siRNA组(0.179±0.017,0.30±0.002,0.296±0.006,0.281±0.007;t=17.802,16.436,14.993,P<0.01);凋亡率较其余各组则明显升高犤(22.28±2.13)%,(4.72±1.67)%,(6.36±0.92)%,(8.19±1.33)%;t=-19.05,-17.31,-15.16,P<0.01犦。③Pavu6+27-黏附斑激酶2转染组bax上升明显,与正常对照组和空质粒转染组比较,差异十分显著(2.15±058,1.01±0.04,1.12±0.04;t=-4.79,-4.318,P<0.01);与阴性对照组比较,差异显著(1.19±0.31,t=-3.59,P<0.05)。④Pavu6+27-黏附斑激酶2转染组B细胞淋巴瘤-2表达较正常对照组、空质粒转染组、阴性对照组显著上升(1.62±0.15,1.04±0.03,1.09±0.01,1.23±0.04;t=-9.39,-8.701,-6.338,P<0.05)。结论:通过RNA干扰技术沉默心脏成纤维细胞中黏附斑激酶基因的表达,可诱导心脏成纤维细胞凋亡,抑制或降低心脏成纤维细胞中黏附斑激酶表达可能是防治心肌纤维化的一种手段。
AIM： To inhibit focal adhesion kinase （FAK） of cardiac fibroblasts in adult rats with RNA interference （RNAi） technique mediated by RNAi vector, and observe changes of cell proliferation and apeptosis. METHODS： The experiment was conducted in the Central Experimental Laboratory of Xinqiao Hospital, Third Military University of Chinese PLA from December 2004 to December 2005. Twenty-four Wistar rats aged 10-12 weeks of clean grade were chosen. Cardiac fibroblasts of reconstructed RNAi plasmid of transfected target FAK were taken as interventional group, untransfected cells, transfected empty plasmids and nonspecific siRNA cells were taken as different control groups. There were totally 4 groups with 6 rats in each group. Cardiac fibroblasts of rats were cultured with digesting method, RNAi vector of FAK were designed, constructed and verified. Inhibiting efficiency of FAK were checked by western blotting method. Cellproliferation was detected with MTr colorimetric assay; Apoptosis percentage was inspected with terminal deoxynucleotidy transferase-mediated nick and labelling （TUNEL）,changes of bcl-2 and bax were measured with flow cytometry. RESULTS： A total of 24 Wistar rats all entered the final analysis.（1） Restriction enzyme analysis and sequencing indicated ＇that RNAi vector of FAK was successfully constructed. Results of Western blot suggested that RNA inhibiting efficiency of RNAi vector of the second reconstructed FAK was the highest, which was 72.1%, others were respecitvely 66.2 % and 55.3 %. （2）A490 value of Pavu6＋27-FAK2 in the transfection group was obviously lower than that in untransfeced cells, transfected empty plasmid and nonspecific siRNA group （0.179 ±0.017,0.30 ±0.002,0.296 ±0.006, 0.281±0.007;t=17.802,16.436,14.993,P 〈 0.01）; And its apoptotic rate was higher than that in other groups [（22.28±9.13）%,（4.72±1.67）%,（636±0.92）%, （8.19±1.33）%;t=19.05,-17.31,-15.16,P 〈 0.01]. （3）Bax in Pavu6＋27-FAK2 transfection group remarkably rised and there were significant differences in comparison with normal control group and empty-plasmid transfection group.（2.15±058,1.01 ±0.04,1.12±0.04; t=-4.79,-4.318,P 〈 0.01）;Compared with negative control group, differences were remarkable（1.19±0.31, t=-3.59, P〈 0.05）. （4）Expression of bcl-2 in Pavu6＋27-FAK2 in transfection group was markedly increased than that in the normal control group, empty-plasmid transfection group and negative control group （1.62 ±0.15,1.04±0.03,1.09±0.01,1.23±0.04 ;t=-9.39,-8.701, -6.338 ,P 〈 0.05 ）. CONCLUSION： Pavu6＋27-FAK siRNA expression vector can inhibit the expression of FAK in cardiac fibroblasts and induce anoikis. Inhibition or reduction of FAK expression in cardiac fibroblasts may be a mean of preventing myocardial fibrosis.
出处
《中国临床康复》
CSCD
北大核心
2006年第25期43-45,共3页
Chinese Journal of Clinical Rehabilitation
基金
国家自然科学基金资助(30100063)~~
