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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 study the inhibitory effect of antisense RNA on the malignant phenotype of human poorly differentiated nasopharyngeal carcinoma CNE-2 cells by using the strategy of inhibiting target gene expression by down-regulating EGFR expression. Methods The 1.35 kb fragment of human EGFR was reversely constructed into the retroviral expression vector pLXSN and transfected into human nasopharyngeal carcinoma CNE-2 cells by lipofectamine. The positive clones were screened and separated by G418. Two The cloned cells transfected with the EGFR antisense cDNA were named CNE-2 / AS4 and CNE-2 / AS8, while the empty vector transfected cells were named CNE-2 / pLXSN. 125I-EGF ligand binding analysis of EGFR expression on the cell membrane, trypan blue staining to measure cell growth changes, and soft agar colony formation assay was used to detect cell transformation, and finally, the selected positive clonal cells were injected into the skin of nude mice The changes of tumor growth inhibition and tumor metastasis were observed at different times. Results The results of ligand binding assay showed that the number of EGFR on the two selected CNE-2 / AS4 and CNE-2 / AS8 cells decreased by 18% and 45%, respectively, compared with that of untransfected cells, indicating that EGFR antisense RNA While the expression of EGFR on the cell membrane was down-regulated. Meanwhile, the growth of CNE-2 cells and the ability of soft agar of EGFR antisense RNA were also significantly lower than that of the control group. After injection into the skin of nude mice, the positive clone cells expressing EGFR antisense RNA showed slow tumor growth and significantly decreased lymph node and lung metastasis. Conclusion These results suggest that CNE-2 cells transfected with EGFR antisense cDNA can downregulate EGFR overexpression and partially inhibit the malignant phenotype of NPC. This is to further elucidate the functional role of EGFR in the occurrence and evolution of nasopharyngeal carcinoma Provided a useful tool