马钱子碱诱导人葡萄膜黑色素瘤细胞凋亡影响及作用机制研究

The study of brucine induces apoptosis of human uveal melanoma cells

目的观察马钱子碱对人葡萄膜黑色素瘤细胞凋亡的影响及作用机制。方法将体外培养的人葡萄膜黑色素瘤细胞分为4组:对照组(CG)、马钱子碱低(BLG)、中(BMG)、高浓度组(BHG),CCK-8法测定细胞增殖率,苏木精-伊红(HE)染色法观察马钱子碱对黑色素瘤细胞形态的影响,Hoechst 33258核染色法观察细胞核凋亡,Annexin V/PI染色检测细胞凋亡率,Western blot法检测黑色素瘤细胞内Bax及Bcl-2蛋白表达。结果(1)细胞增殖率:与CG组比较,BLG组、BMG组及BHG组细胞增殖率均下降,差异有统计学意义(tBLG=7.964,P_(BLG)=0.000;t_(BMG)=12.920,PBMG=0.000;t_(BHG)=20.962,P_(BHG)=0.000);与BLG组比较,BHG组差异有统计学意义(t=10.720,P=0.000);与BMG组比较,BHG组差异有统计学意义(t=6.291,P=0.000)。(2)细胞凋亡率:与CG组比较,BLG组、BMG组及BHG组细胞凋亡率逐渐增加,差异有统计学意义(t_(BLG)=7.982,P_(BLG)=0.000;tBMG=12.744,P_(BMG)=0.000;t_(BHG)=15.960,P_(BHG)=0.000);与BLG组比较,BHG组差异有统计学意义(t=6.940,P=0.000)。(3)Bax蛋白表达:与CG组比较,各组间差异有统计学意义(t_(BLG)=4.082,P_(BLG)=0.004;t_(BMG)=16.740,P_(BMG)=0.000;t_(BHG)=28.990,PBHG=0.000)。与BLG组比较,BMG组差异有统计学意义(t=12.660,P=0.000),BHG组差异有统计学意义(t=23.680,P=0.000)。与BMG组比较,BHG组差异有统计学意义(t=11.020,P=0.000)。(4)Bcl-2蛋白表达:与CG组比较,各组间差异有统计学意义(t_(BLG)=6.678,P_(BLG)=0.000;tBMG=9.080,PBMG=0.000;t_(BHG)=10.079,P_(BHG)=0.000)。与BLG组比较,BMG组差异有统计学意义(t=10.340,P=0.000),BHG组差异有统计学意义(t=14.550,P=0.000)。与BMG组比较,BHG组差异有统计学意义(t=3.674,P=0.006)。结论马钱子碱通过改变线粒体凋亡途径中凋亡相关信号分子Bax及Bcl-2的表达,抑制黑色素瘤细胞增殖并促进细胞凋亡。

OBJECTIVE To observe the effect of brucine on apoptosis of human uveal melanoma cells and its mechanism.METHODS The cultured human uveal melanoma cells were divided into control group(CG),brucine low concentration group(BLG),brucine medium concentration group(BMG)and brucine high concentration group(BHG)groups.The proliferation rate was determined by CCK-8 method,and the effect of brucine on the morphology of melanoma cells was observed by hematoxylin eosin staining(HE),the apoptosis rate was detected by annexin V/PI staining,the expression of Bax and bcl-2 protein in melanoma cells was detected by Western blot.RESULTS(1)Cell proliferation rate:compared with CG group,the cell proliferation rate of BLG group,BMG group and BHG group were decreased,and the difference was statistically significant(tBLG=7.964,PBLG=0.000;t_(BMG)=12.920,P_(BMG)=0.000;t_(BHG)=20.962,P_(BHG)=0.000).Compared with BLG group,there was significant difference in BHG group(t=10.720,P=0.000).Compared with BMG group,there was significant difference in BHG group(t=6.291,P=0.000).(2)Apoptosis rate:compared with CG group,the apoptosis rate of BLG group,BMG group and BHG group increased gradually,the difference was statistically significant(tBLG=7.982,P BLG=0.000;t_(BMG)=12.744,P_(BMG)=0.000;t_(BHG)=15.960,P_(BHG)=0.000).Compared with BLG group,there was significant difference in BHG group(t=6.940,P=0.000).(3)Bax protein expression:compared with CG group,the difference was statistically significant(tBLG=4.082,PBLG=0.004;t_(BMG)=16.740,P_(BMG)=0.000;t_(BHG)=28.990,P_(BHG)=0.000).Compared with BLG group,there was significant difference in BMG group(t=12.660,P=0.000)and BHG group(t=23.680,P=0.000).Compared with BMG group,there was significant difference in BHG group(t=11.020,P=0.000).(4)Bcl-2 protein expression:compared with CG group,the difference was statistically significant(t_(BLG)=6.678,P=0.000;t_(BMG)=9.080,P=0.000;t_(BHG)=10.079,P=0.000).Compared with BLG group,there was significant difference in BMG group(t=10.340,P=0.000)and BHG group(t=14.550,P=0.000).Compared with BMG group,there was significant difference in BHG group(t=3.674,P=0.006).CONCLUSIONS Brucine could inhibit the proliferation and promote apoptosis of melanoma cells by changing the expression of Bax and Bcl-2 in mitochondrial apoptosis pathway.