血管细胞黏附分子-1对卵巢癌细胞凋亡的影响及机制研究

目的:探讨过表达血管细胞黏附分子-1(vascular cell adhesion molecule-1,VCAM-1)对卵巢癌细胞凋亡的影响。方法:构建过表达VCAM-1的慢病毒载体GV358-VCAM1+,转染人类卵巢癌IGROV1细胞株,利用嘌呤霉素筛选稳定表达VCAM-1的IGROV1细胞,通过倒置荧光显微镜下观察绿色荧光,确定细胞转染效率,Western blot及RT-PCR法确定卵巢癌细胞VCAM-1蛋白和m RNA水平;采用流式细胞仪检测过表达VCAM-1的IGROV1的细胞凋亡变化,western blot法检测凋亡相关蛋白(Bcl-2、Bax、Casepase-3、Cleaved Casepase-3)以及STAT3、p-STAT3蛋白表达水平的变化。结果:成功构建的慢病毒载体GV358-VCAM1+在IGROV1细胞中的转染效率达到85%以上,转染细胞的VCAM-1蛋白及m RNA水平均呈稳定表达;VCAM-1过表达卵巢癌细胞的细胞凋亡显著高于空载体对照组(P=0.0149);Bax、Casepase-3、Cleaved Casepase-3表达水平均较对照组显著升高(P0.01),Bcl-2、p-STAT3表达水平明显低于对照组(P0.01),但STAT3表达水平无显著改变。结论:VCAM-1可能通过下调STAT3的磷酸化水平诱导卵巢癌细胞凋亡。

Effect and Mechanism of Vascular Cell Adhesion Molecule-1 on Cell Apoptosis of Ovarian Carcinoma

ABSTRACT Objective: To investigate the effect of the overexpression of vascular cell adhesion molecule-1 on the apoptosis of ovarian carcinoma cells. Methods: The lentiviral vector with full length VCAM-1 gene (GV358-VCAM1+) was constructed and trans- fected into human ovarian carcinoma cell line, IGROV1. The tumor cells with VCAM-1 stable overexpression were selected by puromycin. The transfection rate was determined via the observation of green fluorescence under an inverted fluorescence microscope. The levels of VCAM-1 protein and mRNA were identified by western blot and RT-PCR analysis, respectively. The cell apoptosis was an- alyzed by flow cytometry assay. Meanwhile, the expression level of apoptosis related proteins (Bcl-2, Bax, Casepase-3 and Cleaved Casepase-3), STAT3 and p-STAT3 protein, were detected by western blot analysis. Results: The transfection rate of GV358-VCAM1+ in IGROV1 cells was more than 85%. These cells had stable VCAM-1 expression not only at the protein but also mRNA level. Tumor cells with VCAM-1 overexpression had significantly higher apoptosis than those with empty vector control group (P=0.0149). Moreover, compared with the empty vector control group, there were significantly higher expression of Bax, Casepase-3 and Cleaved Casepase-3(P<0.01), and lower expression of Bcl-2 and p-STAT3 in VCAM-1 overexpression group(P<0.01). However, the expression level of STAT3 was not significantly changed. Conclusion: VCAM-1 could induce the cell apoptosis of ovarian carcinoma through down-regula- tion of STAT3 phosphorylation.