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目的确定端粒酶调节相关hALP基因对细胞DNA损伤的反应和作用。方法以DNA损伤剂H2O2和顺铂处理人HeLa、Hep2细胞,运用定量逆转录聚合酶链反应和免疫荧光观察hALP基因表达的改变,并以荧光素酶报告基因法测定hALP基因启动子活性变化。通过四甲基偶氮唑盐(MTT)法分析不同hALP基因表达状态下DNA损伤剂对细胞生长的影响。结果0.2～1.6mmol/LH2O2可诱导hALPmRNA水平升高,当浓度为0.4mmol/L时,hALPmRNA水平最高;以0.4mmol/LH2O2处理细胞6h即可观察到hALPmRNA水平显著升高,并在36h内保持高水平。顺铂处理细胞也可以上调hALP的mRNA表达,并呈一定的剂量和时间依赖性。免疫荧光检测发现:在0.2或0.4mmol/LH2O2、0.2或0.5μmol/L顺铂处理细胞12h后,hALP的表达水平升高,并且多聚集于细胞核。H2O2和顺铂处理细胞时可通过hALP基因启动子-705～+20nt区域的序列上调其启动子转录活性。H2O2(0.4mmol/L)或顺铂(0.5μmol/L)处理HeLa细胞时,表达正义hALP基因的细胞生长仍活跃,而表达反义hALP基因和对照组细胞则生长相对缓慢。结论DNA损伤可通过激活hALP基因启动子转录活性而上调其表达,hALP基因的表达可以增强细胞对H2O2和顺铂损伤的抵抗性。
Objective To determine the role and response of telomerase-regulated hALP gene to cellular DNA damage. Methods Human HeLa and Hep2 cells were treated with DNA damaging agents H2O2 and cisplatin. The changes of hALP gene expression were observed by quantitative reverse transcription polymerase chain reaction and immunofluorescence. The promoter activity of hALP gene was determined by luciferase reporter assay. The effects of different DNA damage agents on the cell growth were analyzed by MTT assay. Results The level of hALP mRNA was increased when treated with 0.2-1.6 mmol / LH 2 O 2, and the highest level of hALP mRNA was found when the concentration was 0.4 mmol / L. The level of hALP mRNA was significantly increased after treatment with 0.4 mmol / LH 2 O 2 for 6 h, High level. Cisplatin-treated cells also upregulated hALP mRNA expression in a dose- and time-dependent manner. The results of immunofluorescence showed that the expression of hALP was increased at 0.2 or 0.4mmol / LH 2 O 2, 0.2 or 0.5μmol / L cisplatin for 12h, and the expression of hALP was more concentrated in the nucleus. H2O2 and cisplatin treatment of cells by hALP gene promoter -705 ~ +20 nt region sequence upregulation of its promoter transcriptional activity. H2O2 (0.4mmol / L) or cisplatin (0.5μmol / L) treatment of HeLa cells, expression of positive hALP gene cell growth is still active, while the expression of antisense hALP gene and control cells grew relatively slowly. Conclusion DNA damage can up-regulate the transcriptional activity of hALP gene promoter. The expression of hALP gene can enhance the resistance of cells to H2O2 and cisplatin injury.