摘要
目的使用RNA干扰技术阻断人卵巢癌HO-8910细胞中Cyclin E的表达,分析Cyclin E表达受抑制后产生的一些效应,采用双向凝胶电泳和质谱技术分析鉴定转染前后细胞内差异表达的蛋白质,探讨Cyclin E在人卵巢癌细胞增殖中的作用,以期为人卵巢癌发生、发展、诊断与治疗提供有价值的资料。方法构建质粒表达载体pU6-Cyclin E-siRNA,同时设立阴性对照组和空白对照组。采用脂质体转染法稳定转染卵巢癌HO-8910细胞,G418筛选阳性克隆细胞;噻唑蓝(MTT)法检测细胞生长曲线的改变;RT-PCR法比较转染前后Cyclin E mRNA的表达量的变化;双向凝胶电泳-图像分析-质谱技术-数据库搜索,比较转染前后蛋白质组表达的变化,鉴定差异显著的蛋白点。结果 1)成功构建了Cyclin E干扰RNA的重组质粒并获得稳定转染组细胞HO-8910-Cyclin E siRNA。2)稳定转染组HO-8910-Cyclin E siRNA细胞与阴性对照组HO-8910-neo细胞和空白对照组相比,细胞生长速度减慢,Cyclin E mRNA表达水平降低。3)通过双向电泳-图像分析-质谱技术得到稳定转染组低表达蛋白5个,经数据库搜索分别是锌指蛋白ZFP-36、波形蛋白、肿瘤拒绝抗原gp96、未知蛋白、DNA校正蛋白酶Ⅱ。结论Cyclin E干扰RNA干扰后卵巢癌HO-8910细胞Cyclin E基因的表达明显降低,细胞增殖能力减弱。差异表达的蛋白质参与细胞的增殖、细胞信号转导调节,并与肿瘤的发生、发展、诊断密切相关。
cyclin E siRNA were successfully constructed and a stable HO-8910-cyclin E siRNA cell line was obtained.Compared with vector control group （HO-8910-neo cells）and blank control group,cell growth was suppressed and cyclin E mRNA was decreased in HO-8910-cyclin E siRNA cells.Five low-expressed proteins were detected in HO-8910-cyclin E siRNA cells by two-dimensional gel e-lectrophoresis-image analysis-mass spectrometry,which were identified as zinc finger protein 36, vimentin,tumor rejection antigen gp96,unknown protein and DNA correction protease Ⅱ by data-base search.Conclusion Cyclin E siRNA can decrease cyclin E expression and inhibit prolifera-tion in HO-8910 cells.The differentially expressed proteins are involved in the regulation of cell proliferation and signal transduction,and correlated with the occurrence,development and diagno-sis of ovarian cancer.
出处
《南昌大学学报（医学版）》
CAS
2016年第1期41-45,共5页
Journal of Nanchang University：Medical Sciences
