吉西他滨对人子宫颈癌HeLa细胞的放射增敏作用及其机理研究

Mechanistic study of radiosensitization by gemcitabine on human squamous carcinoma cell line of the cervix

目的研究低浓度[即处理24 h时的10％药物致死剂量,24 h IC10]吉西他滨对人宫颈癌HeLa细胞的放射增敏作用及其机理。方法采用四甲基偶氮唑蓝(MTT)法筛选吉西他滨增敏实验的浓度(即24 h IC10);以该浓度吉西他滨处理HeLa细胞4、8、12及24 h,弃药后立刻行60Coγ射线照射(4、6、8 Gy),计数细胞存活分数,计算增敏比;同时采用流式细胞仪分析细胞周期分布和凋亡效应,蛋白印迹杂交法分析p53、bcl-2及box蛋白表达量的变化。结果吉西他滨的24 h IC10,为0.01μmol/L,该浓度吉西他滨分别处理细胞4、8、12及24 h,弃药后立刻照射,增敏比分别为1.19、1.35、1.72、1.93。流式细胞仪分析显示,S期细胞比例随吉西他滨处理时间延长而逐渐升高(P<0.01),其中处理24 h时,S期细胞占51.8％。吉西他滨处理后照射,凋亡细胞比例占21.6％,同时,凋亡相关基因p53、bcl-2及bax蛋白的表达量,与单纯吉西他滨处理及单纯照射相比无明显变化(P>0.05)。结论低浓度吉西他滨对HeLa细胞具有放射增敏作用,其增敏比随吉西他滨处理时间延长而增加。增敏机理与细胞S期阻滞密切相关,而与引发细胞凋亡关系不明显。

Objective To investigate whether gemcitabine ( dFdC ) at the non-cytotoxic concentration enhances the effect of irradiation on human squamous carcinoma cells of the uterine cervix (HeLa) in vitro, and to evaluate the mechanism by which dFdC at the non-cytotoxic concentration[24 h 10% inhibiting concentration ( IC 10 )] is able to enhance radiation-induced cytotoxicity to HeLa in vitro. Methods The non-cytotoxic concentration (24 h IC10 ) of dFdC was determined by methyl thiazolyl tetrazolium(MTT). After exposure to the non-cytotoxic concentration(0. 01 μmol/L) for 4,8,12 and 24 hours followed by immediate irradiation(4,6 and 8 Gy) , the surviving fraction was counted and the radiation enhancement ratio ( RER) was evaluated. Cell cycle distribution and apoptosis were analyzed by flow cytometry. The expressions of p53, bcl-2 and bax were studied by western blot. Results After exposure to non-cytotoxic concentration (0. 01 μmol/L) for4,8,12 and 24 hours followed by immediate irradiation, the RER was 1. 19,1. 35,1. 72 and 1. 93, respectively. After exposed to dFdC, HeLa cells showed an S phase block. The proportion of S phase cells was elevated with the increase of exposure duration (P <0. 01). The S-phase proportion increased to 51. 8% at 24 hours of exposure. Meanwhile, compared with the single-agent treatments, combination of dFdC and radiation did not additionally increase the number of apoptotic cells and expression of proteins related to apoptosis such as p53, bcl-2 and bax ( P > 0. 05). Conclusions HeLa cells were radiosensitive at IC10 concentration of dFdC. The radiosensitization effect depends on the exposure duration to dFdC. There appears a strong association between the radiosensitization and the progression of cells into S-phase after dFdC treatment. Combination of dFdC and radiation did not increase apoptosis of HeLa cells.