吉西他滨对人膀胱癌细胞株T24增殖、凋亡及自噬相关蛋白表达的影响

Effects of gemcitabine on proliferation, apoptosis and expression of autophagy-related proteins in human bladder cancer cell line T24

目的观察吉西他滨对人膀胱癌细胞株T24增殖、凋亡及自噬相关基因表达的影响。方法将T24细胞分为阳性对照组、低剂量实验组、中剂量实验组、高剂量实验组和空白对照组;阳性对照组用10^-8 mol/L紫杉醇处理,低、中、高剂量实验组以1×10^-7,1×10^-8,1×10^-9 mol/L吉西他滨处理,空白对照组不做任何处理。药物干预24,48 h后采用溴化四唑蓝(MTT)法检测细胞增殖情况,流式细胞术检测细胞凋亡情况,蛋白免疫印迹法(WB)检测细胞中自噬相关蛋白表达情况,透射电镜扫描细胞中自噬小体的形成情况。结果与阳性对照组比较,低、中剂量实验组培养24,48 h增殖抑制率(PIR)均明显降低(P均<0.05);实验组T24细胞PIR均随着药物浓度的增加和培养时间的延长而增高(P均<0.05)。阳性对照组和低、中、高剂量实验组细胞凋亡率均显著高于空白对照组(P均<0.05)。随着培养时间的延长,中剂量实验组自噬相关蛋白蛋白表达量呈逐渐升高趋势(P<0.05),P62蛋白表达量呈逐渐减低趋势(P<0.05)。培养4 h后,中剂量实验组细胞中出现了较多的自噬小体。结论吉西他滨可抑制T24细胞增殖,并促其凋亡,具有一定时间-剂量依赖性;同时其还可诱导细胞自噬的发生。

Objective It is to observe the effects of gemcitabine on proliferation,apoptosis and expression of autophagy-related genes in human bladder cancer cell line T24.Methods T24 cells were divided into positive control group,low/medium/high dose experimental groups and blank control group.The positive control group was treated with 10^-8 mol/L paclitaxel,and the low/medium/high dose experimental groups were treated with 10^-7,10^-8,10^-9 mol/L gemcitabine,the blank control group was given no treatment.After 24 and 48 hours of drug intervention,the cell proliferation was detected by MTT assay,apoptosis was detected by flow cytometry,and autophagy-related protein expression was detected by Western blotting(WB),the formation of autophagosomes in cells was observed by transmission electron microscopy.Results Compared with the positive control group,the proliferation inhibition rate(PIR)of the low/medium dose experimental groups were decreased after 24 and 48 hours of culture(P<0.05).The PIR of T24 cells in the experimental group increased with the increase of drug concentration and culture time increased(P<0.05).The apoptotic rate(AR)of the positive control group and the low,medium and high dose experimental groups were significantly higher than that of the blank control group(P<0.05).With the prolongation of culture time,the expression of LC3-Ⅱprotein in the middle dose group increased gradually(P<0.05),and the P62 protein decreased gradually(P<0.05).After 4 hours of culture,more autophagosomes appeared in the cells of the medium dose experiment group.Conclusion Gemcitabine can inhibit the proliferation of T24 cells and promote their apoptosis in a dose-dependent manner,it can also induce autophagy.