Chen Yan, Chen Xiao-yi
Department of Pathology, Guangdong Medical College, Zhanjiang 524023, P. R. China.
Ai Zheng. 2002 May;21(5):498-503.
BACKGROUND & OBJECTIVE: It was proved that Epstein-Brarr virus (EBV) latent membrane protein 1 (EBV-LMP1) has transformation function which plays an important role in the genesis and development of nasopharyngeal carcinoma (NPC) and this function may be associated with apoptosis. This study was designed to investigate the effect of EBV-LMP1 on apoptosis of NPC CNE1 cell line and to explore the possible mechanism of the function of EBV-LMP1 in the genesis and development of NPC.
The authors transfected LMP1 eukaryotic expression plasmid carried with the green fluorescent protein (GFP) reporter gene into CNE1 cell line by electroporation. The expression of GFP was detected by fluorescent microscope; the expression of aimed gene LMP1 was detected by immunohistochemistry and Western blot; the effect of LMP1 on apoptosis of CNE1 cells which were treated either with dexamethasone sodium phosphate or serum starvation was investigated by flow cytometry analysis, fluorescence staining, and DNA fragmentation analysis.
The green fluorescence could be detected in CNE1G cells transfected by control plasmid PAT-GFP and in CNE1GL cells transfected by plasmid PAT-GFP-LMP1, while there was no GFP expression in CNE1 cells untransfected by any plasmid. LMP1 could be detected in CNE1CL cells, but not in CNE1 and CNE1C cells. Apoptosis could be observed in three groups cells which were treated either with dexamethasone sodium phosphate or serum starvation, and the apoptosis index (AI) of CNE1CL cells was higher than that in CNE1 and CNE1G cells (P < 0.05). There were no apoptosis occurring in three group cells when cultured in regular medium.
The LMP1 gene was transfected successfully into CNE1 cells which were stably expressing LMP1. LMP1 could increase apoptosis occurring of CNE1 cells in the process of inducing apoptosis.
已证实爱泼斯坦 - 巴尔病毒(EBV)潜伏膜蛋白1(EBV - LMP1)具有转化功能,在鼻咽癌(NPC)的发生发展中起重要作用,且该功能可能与细胞凋亡有关。本研究旨在探讨EBV - LMP1对NPC CNE1细胞系凋亡的影响,并探索EBV - LMP1在NPC发生发展中作用的可能机制。
作者通过电穿孔法将携带绿色荧光蛋白(GFP)报告基因的LMP1真核表达质粒转染至CNE1细胞系。用荧光显微镜检测GFP的表达;用免疫组织化学和蛋白质印迹法检测目的基因LMP1的表达;通过流式细胞术分析、荧光染色和DNA片段化分析,研究LMP1对经地塞米松磷酸钠处理或血清饥饿处理的CNE1细胞凋亡的影响。
在转染对照质粒PAT - GFP的CNE1G细胞和转染质粒PAT - GFP - LMP1的CNE1GL细胞中可检测到绿色荧光,而在未转染任何质粒的CNE1细胞中无GFP表达。在CNE1CL细胞中可检测到LMP1,而在CNE1和CNE1C细胞中未检测到。在用磷酸地塞米松或血清饥饿处理的三组细胞中均可观察到细胞凋亡,且CNE1CL细胞的凋亡指数(AI)高于CNE1和CNE1G细胞(P < 0.05)。在常规培养基中培养时,三组细胞均未发生凋亡。
LMP1基因成功转染至CNE1细胞,且这些细胞稳定表达LMP1。LMP1可增加CNE1细胞在诱导凋亡过程中的凋亡发生率。