自噬基因Beclin 1在SKOV3细胞中的表达及其调控相关信号传导途径的研究

目的探讨自噬基因Beclin 1在人卵巢癌SKOV3细胞中过表达后自噬相关信号调控通路PI3K/PKB途径中ClassⅠPI3K（p110α）、p-PKB和ClassⅢPI3K（hvps34）的表达变化以及对肿瘤细胞的自噬活性的影响.方法将自噬基因Beclin 1的真核表达载体pcDNA3.1/Beclin1转染SKOV3细胞;分别用荧光定量RT-PCR和Western blot在mRNA和蛋白水平检测用流式细胞仪检测Beclin1、p110α、hvps34和p-PKB的表达;在电镜和荧光显微镜下观察自噬现象,用流式细胞仪检肿瘤细胞自噬情况.结果 pcDNA3.1/Beclin1转染后SKOV3细胞Beclin1表达在mRNA和蛋白质水平明显高于转染空质粒pcDNA3.1和未转染细胞;PcDNA3.1/Beclin1转染后hvps34表达上调,而p110α、p-PKB的表达下调.PcDNA3.1/Beclin1转染SKOV3细胞后在在电镜下可见大量自噬囊泡形成.在荧光显微镜下见pcDNA3.1/Beclin1转染后SKOV3细胞中MDC阳性细胞明显增加.流式细胞仪检测pcDNA3.1/Beclin1转染后SKOV3细胞MDC平均荧光强度高于pcDNA3.1转染组和未转染组.Beclin 1在SKOV3中的过表达后抑制肿瘤细胞在体外的生长,抑制率为58.68%.结论自噬基因Beclin1在人卵巢癌细胞SKOV3中过表达可使ClassⅢPI3K（hvps34）表达上调,ClassⅠPI3K（p110α）及其下游的p-PKB表达下调,诱导肿瘤细胞自噬活性增加,抑制SKOV3在体外的增殖.

Expression of Autophagy Gene Beclin 1 and Involved Signal Transduction Pathway in Ovarian Carcinoma Cell Line SKOV3

Objective To explore the effect of Beclin1 overexpression on the expression of Class I PI3K（p110α）,p-PKB and Class Ⅲ PI3K（hvps34）in PI3K/PKB signaling pathway,and on autophagy in ovarian carcinoma cell line SKOV3.Methods We constructed the recombinant plasmid pcDNA3.1/Beclin1.The vectors were transfected into SKOV3 cells by lipofectamine 2000.The expression levels of Beclin1,hvps34,p110α and p-PKB mRNA and protein were detected by real-time RT-PCR and western blot analysis after transfection.Autophagic vacuoles were observed by electron microscope and fluorescence microscope.Autophagy was measured by flow cytometry（FCM）.MTT was used to evaluate the effect of Beclin1 overexpression on the inhibition of proliferation and growth of the transfected cells and SKOV3 cells.Results Beclin1 mRNA and protein expression were increased in SKOV3 cells transfected with pcDNA3.1/Beclin1.Hvps34 expression was up-regulated and the expression levels of p110α and p-PKB were down-regulated after transfection with pcDNA3.1/Beclin1.A lot of autophagic vacuoles were observed in SKOV3-Beclin1 cells by electron microscope.MDC staining positive cells were increased after transfection with pcDNA3.1/Beclin1.FCM showed that MDC fluorescent intensity of SKOV3 cells transfected with pcDNA3.1/Beclin1 was higher than that of SKOV3 cells transfected with pcDNA3.1 and SKOV3 cells.MTT assay revealed the cell proliferation of SKOV3 cells was inhibited after transfection with pcDNA3.1/Beclin1,and the cell inhibitory rate was 58.68%（P0.05）.Conclusions Beclin1 overexpression can upregulate the expression of Class Ⅲ PI3K（hvps34）,and downregulate the expression of p110α and p-PKB.Beclin1 overexpression can induce autophagy in SKOV3 cells,and inhibit the proliferation of SKOV3 cells in vitro.