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

摘要

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γH2 AX分别由Ho-echst33342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/mp53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BsrI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 p 53 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 cells, 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 H 1299/mp53 cells.Conclusion In tumor cells, 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 p 53 gene expression to affect cell fate .