目的利用反义RNA抑制靶基因表达的策略,下调EGFR的表达而探讨对人类低分化鼻咽癌CNE-2细胞的恶性表型的抑制性效应.方法将人EGFR的N-端1.35kb片段反向构建入逆转录病毒表达载体pLXSN中,并用脂质体介导转染人鼻咽癌CNE-2细胞,G418筛选并分离阳性克隆,将两个EGFR反义cDNA转染的克隆细胞命名为CNE-2/AS4和CNE-2/AS8,而空载体转染的细胞命名为CNE-2/pLXSN.125I-EGF配体结合分析细胞膜上EGFR的表达,台盼蓝染色法测定细胞生长的改变,并用软琼脂集落形成实验检测细胞转化,最后,将筛选的各组阳性克隆细胞分别注射入裸鼠皮下,不同时间观察肿瘤生长的抑制改变及肿瘤的转移状况.结果配体结合实验结果显示,两个选择的克隆CNE-2/AS4和CNE-2/AS8细胞表面EGFR的数量分别较未转染细胞CNE-2下降18%、45%,表明EGFR反义RNA的表达下调了细胞膜上EGFR的表达;同时,EGFR反义RNA表达的CNE-2细胞生长速率和软琼脂生长能力也较对照组细胞明显降低.注射入裸鼠皮下后,EGFR反义RNA表达的阳性克隆细胞表现出肿瘤生长减慢,淋巴结和肺转移能力也明显降低.结论这些实验结果提示EGFR反义cDNA转染的CNE-2细胞能下调EGFR的过量表达并部分抑制鼻咽癌的恶性表型,这为进一步阐明EGFR在鼻咽癌的发生、演化中的功能作用提供了有用的工具.%Objective To determine whether epidermal growth factor receptor (EGFR) expression contributes to tumor growth of poorly differentiated human nasopharyngeal cacinoma CNE-2 cell lines. Methods An expression vector containing a N-terminal fragment (1.35 kb) of human EGFR in the antisense orientation was transfected into CNE-2 cell lines via lipofectamine. The established clones resistant to G418 were isolated and characterized, and the tumor-inhibiting effect of antisense EGFR expression was evaluated in terms of tumor growth and metastasis at different time after subcutanenous inoculation into nude mice. Results Down-regulated EGFR expression in the cells with antisense vector transfection was demonstrated by ligand binding assay. The growth rate and the ability to grow in soft agarose of these antisense transfectants were also reduced.After inoculation into nude mice, EGFR antisense transfectants showed a longer latency period, slower tumor growth and lower metastatic rates to the lymph nodes and lung in comparison with the parental cells. Conclusions These results suggest that these EGFR antisense cDNA-transfected CNE-2 cells are of value to further delineate the role of EGFR in the development and progression ofnasopharyngeal carcinoma.
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