靶向VEGF的shRNA对皮肤鳞癌A431细胞的作用  

Effect of shRNA targeting VEGF on human skin squamous carcinoma cell line A431

作　　者：曹一鑫[1] 冯新[2] 王建力[1] 陈莉[3] 

机构地区：[1]南通大学附属医院皮肤科,江苏南通226001 [2]海门市皮肤病防治医院 [3]南通大学医学院病理学系,江苏南通226001

出　　处：《基础医学与临床》2012年第10期1183-1187,共5页Basic and Clinical Medicine

基　　金：南通市社会发展基金(S2010018);海门市社会事业科技项目计划(H2008-06)

摘　　要：目的在皮肤鳞癌细胞(A431)中下调VEGF mRNA和VEGF蛋白的表达,观察其对A431细胞增殖、迁移和黏附的影响。方法构建psVEGF-shRNA真核表达质粒(psilencer-VEGF1-shRNA,VEGF-s1;psilencer-VEGF2-shR-NA,VEGF-s2)干预A431细胞,同时构建含随机靶序列的阴性对照表达质粒(psilencer-Target-off-shRNA,T-off)。RT-QPCR,Western blot和ELISA检测细胞内VEGFmRNA和蛋白表达;CCK-8法检测细胞活性;流式细胞仪检测细胞周期百分比与细胞凋亡;划痕试验和Transwell小室试验检测细胞二维、三维空间的迁移能力;FN黏附试验检测细胞黏附潜能。结果 A431细胞转染psVEGF-shRNA后活性降低,细胞周期发生阻滞,与对照组相比细胞比率显著增加,在G1期明显降低,在S期伴细胞调亡增加。细胞内VEGFmRNA和VEGF蛋白表达下降,细胞迁移和黏附潜能力均受到抑制(P<0.05)。结论 VEGF是人皮肤鳞癌A431细胞生长相关的重要基因,干扰VEGF基因能有效抑制A431细胞的增殖、迁移和黏附。Objective To inhibit the expression of VEGF mRNA and VEGF protein in the human skin squamous carcinoma A431cell to observe potential affect on proliferation,migration and adhesion.Methods The eukaryotic expression plasmids targeting VEGF（psilencer-VEGF1-shRNA,VEGF-s1;psilencer-VEGF2-shRNA,VEGF-s2） and random target sequence of the negative control plasmid were simultaneously constructed and then transfected these plasmids to A431 cells.The expression of VEGF mRNA and VEGF protein were detected by RT-QPCR,Western blot and ELISA;the vitality of A431 cells was examined by CCK-8 assay;the percentage of cell proliferative cycle and apoptosis of A431cells were tested by flow cytometry;the migration capacity of A431cells in two-dimensional or three-dimensional space were inspected by wound healing effect and transwell assay,respectively;the adhesion potential of A431cells was detected by FN adhesion test.Results After VEGF-s1 or VEGF-s2 treatment,cytoactivity declined,cell cycle arrested,cell proportion increased sharply in G1stage and decreased obviously in S stage with apoptosis increased.The expression of VEGF mRNA and VEGF protein and the migration as well as adhesion of A431 cells were all suppressed significantly,as compared with the control group（P0.05）.Conclusions VEGF is an important growth-related gene of human skin squamous cell carcinoma,silencing VEGF may effectively inhibit the proliferation,migration and adhesion of A431 cells.

关 键 词：发夹状小分子干扰RNA(shRNA) VEGF 人皮肤鳞癌细胞(A431细胞) 

分 类 号：R739.5[医药卫生—肿瘤]