噻吗洛尔对人多发性骨髓瘤细胞株 RPMI 8226增殖、凋亡的影响及其机制

摘要

目的：观察非选择性β-肾上腺素能受体阻断剂噻吗洛尔对人多发性骨髓瘤细胞株RPMI 8226增殖及凋亡的影响，并探讨其可能机制。方法取对数生长期RPMI 8226细胞分为实验1、2、3、4、5、6组，分别加入终浓度为1、2．5、5、20、50、100μmol／L的噻吗洛尔培养，以不添加细胞和药物的培养液为空白对照组，以不添加药物的细胞液为阴性对照组，观察培养24、48、72 h时各组细胞增殖情况。另取适量RPMI 8226细胞，分为A、B、C组，分别加入终浓度为2．5、20、50μmol／L噻吗洛尔培养，设只加细胞培养液的对照组。取培养72 h时各组细胞，观察细胞凋亡情况，检测细胞Bax、B细胞淋巴瘤因子2（Bcl-2）、半胱氨酸天冬氨酸蛋白酶3（Caspase-3）、Caspase-9 mRNA和蛋白表达。结果随浓度升高，细胞增殖抑制率升高，且随干预时间增加，细胞增殖抑制率升高，P均＜0．05。培养72 h时A、B、C组及对照组细胞凋亡率分别为7．17％±0．06％、10．56％±0．65％、15．27％±0．86％、3．93％±0．25％，组间两两比较，P均＜0．05。 A、B、C组Bax mRNA及蛋白的相对表达量均高于对照组（P均＜0．05）。 B、C组Bcl-2 mRNA及蛋白的相对表达量均高于对照组（P均＜0．05）；C组Bcl-2 mRNA相对表达量高于A组（P＜0．05）。 B、C组Caspase-3 mRNA相对表达量高于A组（P均＜0．05）。 A、B、C组Caspase-9 mRNA相对表达量均高于对照组（P均＜0．05）。结论噻吗洛尔可抑制RPMI 8226细胞增殖、促进细胞凋亡，其机制可能与噻吗洛尔可上调Bax、Caspase-3、Caspase-9表达，抑制Bcl-2表达有关。%Objective To investigate the effects of timolol on cell proliferation and apoptosis of human multiple myelo-ma cell line RPMI-8226, and to explore its potential anti-tumor mechanism.Methods The RPMI 8226 cells in the loga-rithmic phase were divided into groups 1, 2, 3, 4, 5, and 6, and then were added with 1, 2.5, 5, 10, 20, 50 and 100μmol/L timolol, respectively.The nutrient solution without adding cells and drugs was taken as the blank control group. The negative control group was only added with cells.At 24, 48 and 72 h of cultivation, MTT method was used to observe the proliferation of different groups.In addition, the appropriate amount of RPMI 8226 cells were divided into groups A, B and C which were added with 2.5, 20 and 50 μmol/L timolol as the final concentrations, respectively.Meanwhile, the control group was only added with cell culture fluid.And then flow cytometry was used to detect the apoptosis effect.Chan-ges in the expression of Caspase-3, Caspase-9, Bax and Bcl-2 protein and mRNA were detected by Western blotting and RT-PCR, respectively.Results After treated with timolol for 24, 48 and 72 h, the inhibition rate increased with the in-crease of drug concentrations and the extension of the time (all P<0.05).After treated with timolol for 72 h, the apoptotic rates of groups A, B, C were 7.17%±0.06%, 10.56%±0.65%, 15.27%±0.86% and 3.93%±0.25% ( all P<0.05).The mRNA and protein expression of Bax in the group A, B and C was significantly high than that of the control group (all P<0.05).The mRNA and protein expression of Bcl-2 in the groups B and C was much higher than that in the control group (all P<0.05).Compared with group A, the mRNA expression of Bcl-2 in the group C was higher.Mean-while, the Caspase-3 mRNA expression was higher in groups B and C as compared with that of group A (all P<0.05). And the mRNA expression of Caspase-9 in the groups A, B and C was higher than that in the control group ( all P<0.05) . Conclusion Timolol inhibits the proliferation and promotes apoptosis of RPMI 8226 cells, the mechanism may be that it can up-regulate the expression of Bax, Caspase-3 and Caspase-9, and inhibit the Bcl-2 expression.