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目的 :研究人类DNA损伤修复基因HR2 4L与细胞凋亡的关系。方法 :PCR方法扩增HR2 4L基因开放阅读框序列 ,克隆入逆转录病毒载体pDOR neo ,转染HeLa细胞 ,筛选阳性克隆 ;Southern印迹验证HR2 4L基因的整合状况。Northern印迹检查HR2 4LmRNA的细胞内表达水平。过氧化氢、丁酸钠及血清饥饿诱导凋亡 ,流式细胞计数仪检测凋亡比例 ,电泳观察DNALadder,Western印迹检测凋亡相关蛋白质的表达。结果 :获得转染有HR2 4L基因的HeLa细胞。与HeLa细胞相比 ,HeLa HR2 4L细胞HR2 4LmRNA表达明显上升。HR2 4L基因过表达导致Bcl 2的表达升高 ,抑制caspase 3和Bax的表达 ,而对p53却无明显的影响。HR2 4L基因过表达抑制过氧化氢和血清饥饿诱导的细胞凋亡 ,但对丁酸钠诱导的细胞凋亡却无抑制作用。结论 :HR2 4L基因过表达抑制细胞凋亡。
Objective: To study the relationship between human DNA damage repair gene HR2 4L and apoptosis. Methods: The open reading frame (ORF) of HR2 4L gene was amplified by PCR, cloned into retrovirus vector pDOR neo, transfected into HeLa cells and screened for positive clones. Southern blot was used to verify the integration of HR2 4L gene. Northern blotting examined the intracellular expression level of HR2 4L mRNA. Hydrogen peroxide, sodium butyrate and serum starvation induced apoptosis. Flow cytometry was used to detect the apoptosis ratio. The expression of apoptosis related protein was detected by electrophoresis and DNALadder, Western blot. Results: HeLa cells transfected with HR2 4L gene were obtained. Compared with HeLa cells, HR2 4L mRNA expression in HeLa HR2 4L cells increased significantly. Overexpression of HR2 4L led to increased expression of Bcl 2, inhibition of caspase 3 and Bax expression, but had no significant effect on p53. Overexpression of HR2 4L inhibited hydrogen peroxide and serum starvation-induced apoptosis but did not inhibit sodium butyrate-induced apoptosis. Conclusion: Overexpression of HR2 4L inhibits apoptosis.