EBV转化B淋巴细胞的差异基因筛选

Scanning of differentially expressed genes of EBV-transformed B lymphocytes

目的通过全基因组芯片筛选正常人外周血淋巴细胞(PBLs)和EB病毒(EBV)转化B淋巴细胞株CGM1中的差异基因,探讨EBV转化B淋巴细胞的机制。方法分离培养正常人外周血淋巴细胞(PBLs),通过原位杂交检测PBLs和CGM1细胞中EBER表达情况;提取2株细胞RNA,利用人全基因组寡核苷酸基因表达谱芯片筛选2株细胞之间差异表达基因,并通过生物信息学对差异表达基因进行GO pathways和KEGG pathways分析。结果 EBER原位杂交结果显示PBLs EBER(-),CGM1 EBER(+);CGM1细胞与PBLs之间差异有统计学意义的基因有1 587个,其中上调基因897个,下调基因690个,这些基因主要涉及P53、PI3K-Akt、Ras、MAPK、Tolllike receptor、WNT、TNF和JAK-STAT等信号通路,与细胞周期调节,B细胞增殖、活化、分化和细胞因子产生,DNA复制、损伤修复,病毒与宿主相互作用及病毒致癌作用等相关。结论 EBV通过细胞周期调节、细胞活化与分化、DNA复制和损伤修复、病毒与宿主相互作用等多个环节参与B淋巴细胞转化。

Objective To investigate the mechanisms of EBV transformation of B cells by scanning the differenti- ally expressed genes between human peripheral blood B cells and the EBV - transformed cell line CGM1. Methods In situ EBER hybridization was performed to separate and culture PBLs from healthy cases and CGM1 ceils. Total RNA was extracted from PBL and EBV - transformed cell, marked with dihydroxyfluorane after reverse transcription, and then hy- bridized through Agilent human whole genome microarray. LIMMA, String, Cytoscape, and other softwares were used to screen and analyze the differentially expressed genes. Results EBER hybridization in situ showed that PBLs were EBV - negative whereas the cell line CGM1 were EBV - positive. Gene expression profile analysis showed a total of 1587 differen- tially expressed genes were screened, including 897 up - regulated and 690 down - regulated genes. These genes were de-termined by GO analysis to be involved in the cell cycle, B cell proliferation, activation, differentiation, cytokine produc- tion, virus infection, DNA repair, DNA metabolic process, viral reproduction, viral carcinogenesis, and virus -host in- teraction. Differentially expressed genes, including 36 key up - regulated genes which primarily control biological proces- ses, such as PI3K- Akt, Ras, MAPK, Toll -like receptor, Wnt, TNF, NF -kappa B, and the Ras and Jak -STAT sig- naling pathways, were determined by KEGG pathway analysis. Conclusion Transformation of human B lymphocytes by EBV is a complicated process involving multiple genes and pathways with virus - host interactions. Gene expression profile analysis showed that EBV may transform human B lymphoeytes by promoting cell cycle and mitosis, inhibiting the cell cy- cle, cell activation, differentiation, DNA repair, and cytokine secretion.