一氧化氮对鼻咽癌细胞放射增敏效应的研究

Radiosensitization effect of nitric oxide on nasopharyngeal carcinoma cells

目的研究外源性一氧化氮(NO)对鼻咽癌5-8F放疗抵抗细胞株(5-8FRs)放射效应的影响,为寻找合适的鼻咽癌放射增敏剂提供实验依据。方法体外培养5-8FRs细胞株,使用不同浓度NO供体药物硝普钠(SNP)干预5-8FRs细胞,CCK-8法检测细胞增殖抑制率筛选出一个对5-8FRs细胞增殖无明显影响的IC01 SNP浓度(细胞增殖抑制率为1%的SNP浓度);使用1、2、4、6 Gy和8 Gy放射线干预5-8FRs细胞,确定IC15放射剂量(细胞增殖抑制率为15%的放射剂量)。用IC01SNP浓度、IC15放射剂量放疗单独干预及二者联合干预5-8FRs细胞,高倍镜下观察细胞形态学变化,CCK-8法检测各分组细胞增殖抑制率,流式细胞仪检测细胞凋亡情况,硝酸还原法检测细胞上清液中NO浓度。结果⑴SNP以浓度依赖方式、放射线以剂量依赖方式抑制5-8FRs细胞的增殖,其中SNP IC01为(513.89±14.69)μmol/L(SNP组);放射剂量IC15为(3.96±0.33)Gy(放疗组);⑵联合组(SNP+放疗)与单独SNP组和放疗组相比,5-8FRs细胞形态学差异显著,漂浮细胞显著增多,贴壁细胞数量逐渐减少并失去原有形态;⑶IC01的SNP浓度对5-8FRs细胞增殖无明显影响,然而联合组较单独放疗组细胞抑制率显著提高(t=7.708,P<0.01);并且联合组中NO浓度显著高于单组放疗组[(310.03±5.76)μmol/L vs(77.34±2.60)μmol/L,P<0.05];⑷5-8FRs自发凋亡率为(1.35±0.06)%,SNP组凋亡率为(2.22±0.37)%,SNP组细胞凋亡无明显变化,放疗组凋亡率为(15.37±0.65)%,联合组为(50.27±2.24)%,联合组较放疗组促凋亡能力显著增强(t=-21.459,P=0.001)。结论合适浓度的外源性NO可在对细胞本身不产生明显毒性的情况下可显著增加5-8FRs细胞株放射敏感性。

Objective To investigate the effects of exogenous nitric oxide(NO)on radiation response of nasopharyngeal carcinoma 5-8F radiotherapy resistant cell line(5-8FRs)and to provide experimental basis for finding suitable radiosensitizer on nasopharyngeal carcinoma.Methods 5-8FRs cells were cultured in vivo and treated with sodium nitroprusside(SNP)of different concentrations.The inhibition rate of cell proliferation was detected by cell counting kit-8(CCK8)method.A concentration of IC01 SNP(1%SNP)which had no obvious effect on the proliferation of 5-8FRs cells was screened out.The 5-8FRs cells were intervened with 1,2,4,6 Gy and 8 Gy radiation to determine the radiation dose of IC15(15%radiation dose).5-8FRs cells were treated with IC01 SNP concentration,IC15 radiation dose and radiotherapy alone or in combination.The morphological changes of 5-8FRs cells were observed under microscope.The proliferation inhibition rate of each group was detected by CCK-8 method,the apoptosis was detected by flow cytometry,and the concentration of NO in cell supernatant was detected by nitrate reduction method.Results⑴The proliferation of 5-8FRs had been inhibited in a concentration-dependent manner by SNP and a dose-dependent manner by radiation.The SNP concentration of IC01 was(513.89±14.69)μmol/L(SNP group).The radiation dose of IC15 was(3.96±0.33)Gy(radiotherapy group);⑵Compared with single SNP group and radiotherapy group,the morphology of 5-8FRs cells in combination group(SNP+radiotherapy)was significantly different,floating cells increased significantly,the number of adherent cells gradually decreased and lost their original morphology;⑶SNP concentration of IC01 had no significant effect on the proliferation of 5-8FRs cells,but the inhibition rate of combined group was significantly higher than that of radiotherapy group(t=7.708,P<0.01);The concentration of NO in the combined group was significantly higher than that in the single radiotherapy group[(310.03±5.76)μmol/L vs(77.34±2.60)μmol/L,P<0.05];⑷The spontaneous apoptosis rate of 5-8FRs was(1.35±0.06)%,while the apoptosis rate in the group of IC01SNP was(2.22±0.37)%,with no significant difference.The apoptosis rate of 5-8FRs in the combined group(50.27±2.24)%was significantly higher than that of the group of IC15 radiation dose(15.37±0.65)%.Conclusions Under no obvious toxicity to cells themselves circumstances,exogenous nitric oxide with appropriate concentration could significantly enhance the radiosensitivity on 5-8FRs.