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[目的]探讨研究腺病毒介导的鸟氨酸脱羧酶反义RNA对肺癌的抑制作用。[方法]选择肺癌细胞A-549,采用人鸟氨酸脱竣酶的基因序列扩增引物和扩增腺病毒基因组E1区的引物,对ODC基因片段的克隆,并构建重组体现病毒载体。测定病毒的滴度,并采用MTT法测定病毒对肺癌细胞A-549的抑制作用。[结果]克隆得到了含起始密码ATG的ODC外显子上的一段基因,并可经序列测定,再将这一段基因反向插入腺病毒载体后,也可通过PCR进行初步的鉴定。另外测得病毒的滴度为2.5×108pfu/ml,且通过MTT法测得该病毒对A-549肺癌细胞随作用时间的延长和病毒浓度的增高,抑制作用越强。[结论腺病毒介导的鸟氨酸酶反义RNA对肺癌具有明显的作用,但对其基因治疗的应用还应通过进一步的研究。
[Objective] To investigate the inhibitory effect of adenovirus-mediated ornithine decarboxylase antisense RNA on lung cancer. [Method] The lung cancer cells A-549 were selected and the primers of human ornithine desaturase gene sequence and primers of E1 region of adenovirus genome were amplified. The ODC gene fragment was cloned and a recombinant virus vector was constructed. The titer of the virus was measured, and the inhibitory effect of the virus on the lung cancer cell A-549 was determined by MTT assay. [Result] A segment of ODC exon gene containing ATG with the initiation codon was cloned and sequenced. The gene was reversely inserted into the adenovirus vector and then identified by PCR. In addition, the titer of the virus was 2.5 × 108pfu / ml, and the inhibitory effect of the virus on the A-549 lung cancer cells was prolonged with the increase of the virus concentration and the virus concentration by MTT assay. [Conclusion] Adenovirus-mediated ornithine antisense RNA has obvious effect on lung cancer, but its application of gene therapy should be further studied.