中文摘要：

目的研究EB病毒核抗原1（Epstein—Barr virus nuclear antigen1，EBNAI）对人鼻咽癌细胞增殖及细胞周期的影响。方法构建特异性的短发夹RNA（short hairpin RNA，shRNA）EBNA1慢病毒干扰载体，包装慢病毒后感染过表达EBNA1的鼻咽癌细胞C666－EBNA1（简写为CE）。应用四甲基偶氮唑蓝法（MTT）和细胞计数法检测细胞增殖状态，流式细胞术、荧光定量PCR及蛋白免疫印迹法检测细胞周期及相关调控因子变化。结果成功构建含shRNA EBNA1的重组慢病毒，其感染可显著下调CE细胞中EBNA1表达。细胞计数和MTF均证明CE—shRNA细胞的增殖能力较CE-对照组显著下降（P值均〈0．05）。细胞周期提示干扰EBNAl后cE细胞G0．G1期比例由（62．43±6．62）％升高到（89．66±0．64）％，两者比较差异有统计学意义（t=-7．091，P=0．002），S期比例由（34．93±7．36）％下降为（7．824-2．44）％，两者比较差异有统计学意义（t=6．095，P=0．004），G2-M期无明显变化（t=0．090，P=0．933）。抑制EBNA1后，c—myc、CDK4、CDK6、pRb的mRNA水平分别下调65．60％、34．06％、41．05％、55．29％。蛋白免疫印迹法证实抑制EBNA1后4种蛋白的表达亦下凋。结论沉默EBNA1基因可抑制鼻咽癌细胞增殖，其机制之一可能是通过影响c—myc、CDK4、CDK6、pRb等基因的表达使鼻咽癌细胞阻滞于G0-G1期。

英文摘要：

Objective To study the effect of Epstein-Barr virus nuclear antigen 1 （EBNA1） on cell proliferation and cell cycle in nasopharyngeal carcinoma （NPC） cells. Methods Recombinant lentivirus that encoded EBNA1 short hairpin RNA （shRNA） was prepared. The C666-EBNA1 （CE） cells were transduced with lentivirus and selected by fluorescence activated cell sorting （FACS） to repress EBNA1 expression. The protein expression levels of EBNAI were examined by Western blot. The effect of EBNA1 silence on cell proliferation was analyzed by MTT assay and cell growth assay, respectively. Cell cycle was assessed by flow cytometry. The mRNA and protein levels of cell cycle regulators were examined by real-time PCR and Western blot. Results Recombinant lentivirus encoded EBNA1 shRNA was successfully constructed. The EBNA1 expression in CE ceils was significantly reduced by lentivirus-mediated RNA interference. The results of cell counting and MTT assay showed that EBNA1 down-regulation significantly inhibited cell growth in CE-shRNA EBNA1 cells （ P 〈 0. 05 ）. Compared with the control group, the percentage of cells in G0-G1 phase was increased from （62. 43 ± 6. 62） % to （ 89.66±0. 64） % （ t = -7. 091, P =0. 002）, and that in S phase was decreased from （34. 93 ± 7. 36）% to （7.82 ± 2.44）% （t =6. 095, P = 0. 004）. The mRNA expressions of c-myc, CDK4, CDK6 and pRb were decreased by 65.60%, 34. 06%, 41.05% and 55.29% respectively with the similar results in protein expression levels. Conclusions Suppression of EBNA1 may inhibit the growth of nasopharyngeal carcinoma cells in vitro and induce a Gl-phase cell cycle arrest, which might be mediated by down-regulation of c-myc, CDK4, CDK6 and pRb.