蒺藜皂苷D促进膀胱癌T24细胞凋亡的作用及机制研究

Effect and mechanism of Terrestrosin D in promoting apoptosis of bladder cancer T24 cells

目的探讨蒺藜皂苷D对膀胱癌T24细胞凋亡的调控作用及其分子机制。方法将膀胱癌T24细胞分为空白组、对照组、低剂量实验组、中剂量实验组、高剂量实验组。空白组不做干预,对照组加入等量50%的乙醇,低、中、高剂量实验组分别加入浓度为30,60,90 mg·L^(-1)的蒺藜皂苷D干预处理。用实时无标记细胞分析技术(RTCA)实时检测细胞生长状况;用流式细胞术实验检测细胞凋亡情况;用蛋白质印迹(Western blot)法检测凋亡抑制蛋白B细胞淋巴2(Bcl-2)及促凋亡蛋白Bcl-2相关蛋白X(Bax)的表达水平。结果细胞存活率分析实验结果显示各浓度的蒺藜皂苷D对膀胱癌T24细胞均有生长抑制作用,48 h时半数抑制浓度(IC50)经检测为35.98 mg·L^(-1)。空白组,对照组,低、中、高剂量实验组细胞72 h的增殖率分别为(241.01±21.43)%,(8.74±0.87)%,(85.73±8.47)%,(45.51±1.42)%和(27.45±2.59)%;干预24 h后的凋亡率分别为(3.81±1.02)%,(29.12±3.10)%,(8.18±2.12)%,(16.77±3.68)%和(26.66±3.15)%。低、中、高剂量实验组与空白组比较,增殖率显著下调,凋亡率显著升高,差异均有统计学意义(均P<0.05)。空白组、对照组、低、中、高剂量实验组的Bcl-2相对表达水平分别为0.61±0.03,0.21±0.06,0.56±0.05,0.38±0.09和0.32±0.05;Bax相对表达水平分别为0.19±0.06,0.62±0.08,0.24±0.08,0.39±0.08和0.53±0.04。上述指标,低、中、高剂量实验组与对照组比较,差异均有统计学意义(均P<0.05)。结论蒺藜皂苷D通过调控膀胱癌T24细胞凋亡关键蛋白Bcl-2及Bax的表达,以此抑制肿瘤细胞生长并促进其凋亡。

Objective To investigate the regulatory effect of Terrestrosin D on the apoptosis of bladder cancer T24 cells and its molecular mechanism.Methods Bladder cancer T24 cells were divided into blank group,control group,experimental-L group,experimental-M group,experimental-H group.The blank group was not intervened;the control group was added with the same amount of 50%ethanol;the experimental-L,-M,-H groups were added with 30,60 and 90 mg·L^(-1) of Terrestrosin D,respectively.Real-time label-free cell analysis technology was used to detect cell growth in real time;flow cytometry was used to detect cell apoptosis;Western blot was used to detect apoptosis inhibitory protein Bcl-2 and the expression level of the pro-apoptotic protein Bax.Results The results of cell viability analysis showed that all concentrations of Terrestrosin D had inhibitory effects on the growth of bladder cancer T24 cells,and the median inhibitory concentration(IC50)was detected at 35.98 mg·L^(-1) at 48 h.The proliferation rates of cells in the blank group,control group,experimental-L,-M,-H groups at 72 h were(241.01±21.43)%,(8.74±0.87)%,(85.73±8.47)%,(45.51±1.42)%and(27.45±2.59)%,respectively;the apoptosis rates after 24 h of intervention were(3.81±1.02)%,(29.12±3.10)%,(8.18±2.12)%,(16.77±3.68)%and(26.66±3.15)%,respectively.Compared with the blank group,the proliferation rates in the experimental-L,-M,-H groups were significantly decreased,and the apoptosis rates significantly increased,the differences were statistically significant(all P<0.05).The relative expression levels of Bcl-2 in the blank group,control group,experimental-L,-M,-H groups were 0.61±0.03,0.21±0.06,0.56±0.05,0.38±0.09 and 0.32±0.05,respectively;the relative expression levels of Bax were 0.19±0.06,0.62±0.08,0.24±0.08,0.39±0.08 and 0.53±0.04,respectively.Compared experimental-M group,experimental-H group with the blank group,the differences of the obove indexes were statistically significant(all P<0.05).Conclusion Terrestrosin D inhibits the growth of tumor cells and promotes their apoptosis by regulating the expression of the key apoptosis proteins of bladder cancer T24 cells induding Bcl-2 and Bax.