摘要
目的 肺癌的发生是多因素作用的结果 ,RON基因参与其中 ,但它在肺癌的基因治疗中意义尚不明确。本研究旨在探讨RON能否作为肺癌的基因治疗靶点及针对该基因跨膜区段 (RONm)反义核酸对肺癌细胞的生物学功能的影响。方法 从人肺鳞癌细胞提取总RNA进行RT PCR、T载体克隆和测序 ,获得RON基因的跨膜区段 ,并将其反向插入真核表达载体中 ,转染肺癌细胞 ,利用ELISA检测细胞模型RON基因表达后的蛋白水平 ,同时利用MTT和细胞记数方法监测细胞的生物学活性的变化。结果 用RT PCR亚克隆RONm ,测序结果与GenBank(X70 0 4 0 )一致。构建的RONm反义真核表达载体 ,转染后的肺癌细胞株A5 4 9RON基因表达量和细胞活性明显降低。结论 成功构建RONm反义核酸真核表达载体 ,RON能作为肺癌的基因治疗靶点 ;RONm反义核酸可有效抑制RON基因的表达阳性肺癌细胞的生长 ,为进一步研究将RONm反义核酸作为一种的肺癌基因治疗方法打下了分子生物学基础。
Objectives To investigate in vitro inhibition of RON gene expression and the effect of anti sense nucleic acid of transmembrane domain of RON gene(RONm) on biological functions of lung cancer cell line A549, and to explore the possibility of the anti sense nucleic acid of RON gene as a candidate for lung cancer gene therapy. Methods Total RNA from lung cancer cells of human squamous carcinoma was extracted. The cDNA encoding RONm was cloned by reverse transcription polymerase chain reaction (RT PCR) and inversely inserted into BamH I and Hind III sites of the eukaryotic expression vector pcDNA3.1,then the anti sense nucleic acid of this domain was transferred into the studied cells, A549. The expression of RON gene in the experimental and control groups were monitored by ELISA and the alteration of cell proliferation was examined by MTT assay and cell count. Results The cDNA encoding RONm was cloned and its sequence accorded with the data from GenBank (X70040). The pcDNA 3.1 vector contained the RONm antisense nucleic acid. The expression level of RON gene in the transfected lung cancer cells was reduced and the cell proliferation was inhibited as compared with the control cell lines. Conclusion The RONm antisense nucleic acid eukaryotic expressing vector was successfully constructed and the anti sense nucleic acid of RONm significantly reduces the expression of RON gene in the highly expressed lung cancer cell line A549 and might inhibit the growth of this cell line.
出处
《中华老年多器官疾病杂志》
2004年第4期283-288,共6页
Chinese Journal of Multiple Organ Diseases in the Elderly
