Skp2基因沉默对大肠癌细胞SW480生物学特性的影响

Effects of Skp2 Gene Silence on Biological Characteristics of Human Colorectal Cancer Cells

目的:观察Skp2基因沉默对人大肠癌SW480细胞增殖与凋亡的影响。方法:通过细菌内同源重组法,构建含有H1启动子驱动、表达绿色荧光蛋白(GFP)标志和Skp2基因干扰片段的腺病毒骨架载体。通过测序鉴定后在HEK293细胞中包装成腺病毒,并扩增纯化。体外感染人大肠癌SW480细胞,在荧光显微镜下观察GFP表达,以确定转染效率;用蛋白免疫印迹技术检测基因沉默效率。用MTT比色法检测细胞生长率,流式细胞技术检测细胞周期和凋亡。结果:琼脂糖电泳和测序鉴定表明针对Skp2基因干扰片断的碱基序列及插入位点完全正确;Skp2基因沉默后影响大肠癌SW480细胞的周期进程,使其发生G0/G1期阻滞,抑制了细胞的增殖,使其生长速度减缓;并促使大肠癌SW480细胞凋亡。结论:Skp2基因沉默后影响大肠癌SW480细胞的细胞周期,发生G0/G1期阻滞,抑制了细胞的增殖,并诱导细胞凋亡。

Objective To investigate the effects of Skp2 gene silence on the proliferation and apoptosis of human colorectal cancer cells. Methods The siRNA containing DNA sequence targeting against Skp2 or its non-specific sequence were synthesized and subcloned into shuttle-H1 vector that containing green fluorescent protein( GFP) DNA sequence. The subcloning was confirmed by DNA sequencing. The shuttle plasmid was linearized and recombined with back-bone pAdEasy-1 in BJ5183 bacteria. The recombinant adenovirus-Skp2-siRNA particles were produced by transfection of HEK293 cells. The recombinant adenovirus was packaged and amplified in HEK293 cells with high purity and high titer. SW480 cells were infected by recombinant adenovirus in vitro,the expression of GFP was observed under fluorescence microscope,so as to confirm the efficiency of transfection. The level of Skp2 protein was detected by western blotting. The cell cycle and cell apoptosis was assessed by flow cytometry. The proliferation of cells was analyzed by MTT assay. Results The results of agarose electrophoresis and DNA sequencing demonstrated that the base sequence and insertion site of interference fragment targeting against Skp2 gene were absolutely correct. The cell cycle of SW480 cells arrested at G0/ G1phase,the proliferation and growth of cells was inhibited,even the apoptosis was induced by Skp2 gene silence. Conclusion Skp2 gene silence can affect the cell cycle,induce G0/ G1phase arrest,inhibit cell proliferation,induce apoptosis of colorectal cancer SW480 cells.