摘要
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。
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 lipofec-tamine. 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 lig-and 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 low-er 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 of nasopharyngeal carcinoma.
出处
《第一军医大学学报》
CSCD
北大核心
2003年第9期877-881,共5页
Journal of First Military Medical University
