RNAi沉默RhoC基因对人脐静脉内皮细胞体外生物学行为的影响  

Influence of RNA interference-induced RhoC gene silencing on behaviors of human umbilical vein endothelial cells

作　　者：赵良平[1] 张庆华[1] 石晓燕[1] 韩志强[2] 熊国平[1] 黄磊[1] 田训[1] 

机构地区：[1]华中科技大学同济医学院附属武汉中心医院妇产科,武汉430014 [2]华中科技大学同济医学院附属同济医院妇产科

出　　处：《中华实验外科杂志》2012年第9期1713-1715,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81101950）

摘　　要：目的观察RNA干扰（RNAi）沉默RhoC基因表达对人脐静脉内皮细胞（HUVEC）生物学行为的影响。方法构建RhoC基因特异性短发夹状RNA（shRNA）真核表达载体，以脂质体介导转染HUVEC，采用逆转录一聚合酶链反应（RT—PCR）和Westernblot法检测RNAi后细胞中RhoCmRNA及蛋白表达水平变化；划痕试验检测细胞迁移能力，噻唑蓝（MTr）比色法测定转染前后细胞增殖及黏附能力的变化。胶原基质胶三维培养检测内皮细胞体外血管生成能力。结果转染RhoCshRNA的实验组与空载体对照组比较，RhoCmRNA的表达分别为（10．93±4．72）％和（56．77±13．79）％，蛋白质的表达分别为（19．95±7．31）％和（62．06±22．75）％，细胞愈合百分比分别为（45．08±12．93）％和（73．49±10．83）％，增殖能力[吸光度（A）值]分别为1．002±0．283和1．629±0．192，血管样结构分别为2．85±1．08和4．46±1．38，而黏附能力（A值）分别为0．795±0．097和0．907±0．178。实验组较对照组迁移能力、增殖能力和血管样结构生成能力显著降低（P〈0．05），转染后细胞的体外黏附能力无明显变化（P〉0．05）。结论抑制RhoC表达能降低HUVEC体外增殖、迁移及血管生成能力。Objective To investigate the effects of RhoC gene silencing by RNA interference （RNAi） on behaviors of human umbilical vein endothelial cells （HUVECs） in vitro. Methods The vector containing shRNA targeting RhoC gene was constructed and transfected into HUVECs by Lipofeetamine- 2000. The expression of Rhoc mRNA and protein in HUVECs was detected respectively by reverse tran- seription-polymerase chain reaction （ RT-PCR ） and Western blotting. Wound-healing assay in vitro and three-dimensional cultures were used to evaluate the migration and angiogenesis capacity of HUVECs. The changes in the adhesion and proliferation of the cells were examined by methyl thiazol tetrazolium （MT＇F） assay. Results In RhoCshRNA-transfected group and control group, the expression of RhoC mRNA and protein was （10.93_±4.72）% and （19.95 ±7.31）% , and （56.77 ±13.79）% and （62.06±22.75）% respectively, and the per-cent wound closure was （45.08 ± 12. 93 ）% and （73.49 ± 10. 83 ）% respective- ly. The absorbance （A） value of proliferation was 1. 002 ± 0. 283 and 1. 629 -± 0. 192. The number of cordlike was 2. 85 ± 1.08 and 4.46 ± 1.38, and the adhesion vasue was 0. 795 ± 0. 097 and 0. 907 ± 0. 178. The expression of RhoC was suppressed by transfecting RhoCshRNA into HUVEs, meanwhile, the migration, proliferation and angiogenesis capacity of HUVECs were decreased. No significant difference was found in the adhesion of the cells between RhoCshRNA-transfected group and control group. Conclu- sion RNAi-induced RhoC gene silencing may decrease the capacity of HUVE migration, proliferation and angiogenesis in vitro.

关 键 词：RNA干扰 RHOC 人脐静脉内皮细胞 细胞迁移 血管形成 

分 类 号：R730.2[医药卫生—肿瘤]