一氧化氮对辐射诱导的肿瘤细胞凋亡和DNA双链断裂的双向性影响

Dual effects of nitric oxide on radiation induced cellular apoptosis and DNA double strand breaks in tumor cells

目的观察不同浓度的一氧化氮在不同p53基因状态的肿瘤细胞中对射线导致的细胞凋亡和DNA双链断裂的双向性影响。方法 2009年6月—2010年6月于第四军医大学放射医学实验室进行实验。选用p53缺如人非小细胞肺癌H1299细胞,稳定转染野生型p53(wtp53)或突变型p53(mp53)基因,得到H1299/wtp53或H1299/mp53细胞。细胞内质粒载体的稳定表达由RT-PCR和限制性内切酶片段长度多态性分析(RFLP)法检测。细胞先于含不同浓度硝酸异山梨酯(ISDN)的培养基中培养,然后接受X线照射。照射后细胞凋亡和γH2AX分别由Hoechst33342染色法和流式细胞术测定。Western blot检测凋亡相关蛋白的表达。结果在H1299/wtp53和H1299/mp553细胞中,在p53基因7号外显子RT-PCR的产物中可观察到清晰的110 bp条带。p53基因7号外显子用MspI或Bsr1限制性内切酶特异性消化后,相应地在H1299/wtp53或H1299/mp53细胞中,可以观察到清晰的70 bp条带。在H1299/wtp53细胞中,低浓度的ISDN(5μmol/L)可显著降低X线辐射诱导的凋亡和DNA双链断裂,而高浓度的ISDN(500μmol/L)却显著升高X线辐射诱导的凋亡和DNA双链断裂。而且,一氧化氮对辐射导致的Caspase-3和Bax介导的凋亡有双向性的影响。但是,对于上述浓度的ISDN,并没有在H1299/mp53细胞中观察到这些现象。结论在肿瘤细胞中,不同浓度的一氧化氮可双向性影响辐射导致的Bax和Caspase-3介导的凋亡以及DNA双链断裂的变化,并且一氧化氮可通过p53基因表达调控来影响细胞的命运。

Objective To investigate the effects of different concentrations of nitric oxide （NO） on apoptosis and DNA double strand breaks in tumor cells with different p53 gene status. Methods The experiment was carried out in the labo- ratory of radiation medicine, The Fourth Military Medical University from June 2009 to June 2010. The p53 of human non- small cell lung cancer H1299 cells, stably transfected with wild-type or mutant p53 （wtp53） p53 （mp53） gene, H1299/ wtp53 or H1299/mp53 cells. Analysis by RT-PCR and restriction fragment length polymorphism expression vectors in the cells was stable （RFLP） detection method. The cells were cultured in medium containing different concentrations of isosorbide dini- trate （ISDN）, and then received X-ray irradiation. After irradiation, apoptosis and gamma H2AX were measured by Ho- echst33342 staining and flow cytometry, respectively. The expression of apoptosis related protein was detected by Western blot. Results In H1299/wtp53 and H1299/mp53 cells, observed a clear band of 110 bp RT products can be PCR in exon 7 of the p53 gene. Exon 7 of p53 gene with MspI or BsrI restriction endonuClease digestion specificity, corresponding in H1299/ wtp53 or H1299/mp53 ceils, can clearly observe the band of 70 bp. In H1299/wtp53 cells, the low concentration of ISDN （5 μmol/L） can significantly reduce the X-ray radiation induced apoptosis and DNA double strand breaks, whereas high con- centrations of ISDN （500 μmol/L） was significantly increased apoptosis and DNA X-ray radiation induced double strand breaks. Furthermore, nitric oxide has bidirectional effects on radiation-induced Caspase 3 and Bax mediated apoptosis. Howev- er, for these concentrations of ISDN,none of these phenomena were observed in H1299/mp53 cells. Conclusion In tumor ceils,changes of nitric oxide at different concentrations can be dual effects of radiation induced Bax and Caspase 3 mediated apopto- sis and DNA double strand breaks, and nitric oxide through the regulation of p53 gene expression to affect cell fate.