摘要
目的探讨爱泼斯坦-巴尔病毒(Epstein-Barr virus,EBV)编码的BARF1基因对人胃上皮细胞(GES-1)增殖、迁移及细胞周期分布的影响。方法将pIRES-EGFP/BARF1重组质粒、pIRES-EGFP空载体转染GES-1细胞,通过G418筛选,获得稳定表达pIRES-EGFP/BARF1及pIRES-EGFP的GES-1细胞。在荧光显微镜下观察转染与未转染组细胞生长状况;通过逆转录PCR(Reverse Transcription-PCR,RT-PCR)检测转染细胞中BARF1基因表达情况;通过细胞计数及CCK8法评估转染与未转染三组细胞的增殖能力;通过免疫组织化学(Immunohistochemistry,IHC)染色法评价不同组别细胞周期蛋白Cyclin D1的表达情况;应用划痕实验分别对稳定转染BARF1组和空载体组GES-1细胞的迁移效率进行分析评价;应用流式细胞仪检测与分析细胞周期的分布情况。结果获得稳定表达pIRES-EGFP/BARF1、pIRES-EGFP的GES-1细胞模型;稳定转染BARF1组的细胞增殖能力高于未转染组及转染空载体组,并且稳定转染BARF1组S期显著延长;稳定转染BARF1组GES-1细胞的迁移能力比转染pIRES-EGFP空载体组得到明显提升;细胞周期蛋白Cyclin D1集中表达在稳定转染BARF1组的GES-1细胞核内。结论作为EBV编码的癌基因BARF1能够促进人胃上皮细胞GES-1增殖、迁移及分裂,抑制细胞凋亡,促进胃上皮细胞的恶性转化。
The effects of BARF1 gene encoded by Epstein-Barr virus on proliferation,migration and cell cycle distribution of human gastric epithelial cells(GES-1)were investigated.GES-1 cells were transfected with recombinant plasmid of pIRES-EGFP/BARF1 and empty vector of pIRES-EGFP.GES-1 cells with stable expression of pIRES-EGFP/BARF1 and empty vector of pIRES-EGFP were obtained by G418 screened.The GES-1 cell growth of transfected with pIRES-EGFP/BARF1 and pIRES-EGFP,and untransfected were observed under fluorescence microscope.The expression of BARF1 gene in the transfected cells was detected by reverse transcription-PCR(RT-PCR).The proliferation of transfected and untransfected cells was evaluated by cell count and CCK8 method.The expression of CyclinD1 in different groups was evaluated by immunohistochemical staining.The migration efficiency of GES-1 cells stably transfected with BARF1 and empty vector was analyzed and evaluated by wound-healing assay.The cell cycle distribution was detected and analyzed by flow cytometry.The GES-1 cell model stably expressing pIRES-EGFP/BARF1 and pIRES-EGFP was obtained;the proliferation ability of cells in the stable transfected BARF1 group was higher than that of the untransfected group and the transfected empty vector group,and the S phase of the stable transfected BARF1 group was significantly prolonged.The migration ability of GES-1 cells stably transfected with BARF1 was significantly higher than that of pIRES-EGFP emptyvector group.CyclinD1 was concentrated in the nucleus of GES-1 stably transfected BARF1 group.BARF1,an oncogene encoded by EBV,can promote the proliferation,migration and division of human gastric epithelial cells GES-1,inhibit apoptosis and promote the malignant transformation of gastric epithelial cells.
作者
杨佳宁
李娜
董鸿铭
徐媛
李淑英
YANG Jia-ning;LI Na;DONG Hong-ming;XU Yuan;LI Shu-ying(North China University of Science and Technology/Hebei Key Laboratory for Chronic Diseases,Tangshan 063210,China)
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2021年第12期1071-1077,1083,共8页
Chinese Journal of Zoonoses
基金
河北省留学人员科技活动资助项目(No.CY201620)。
