苦参碱对葡萄膜黑色素瘤细胞增殖、凋亡及放射敏感性的影响

Effect of matrine on proliferation,apoptosis and radiotherapy sensitivity of uveal melanoma cells

目的探讨苦参碱对葡萄膜黑色素瘤细胞增殖、凋亡及放射敏感性的影响。方法实验研究。体外实验:对数生长期人脉络膜黑色素瘤MuM2B细胞随机分为对照组和苦参碱0.25、0.50、1.00、2.00 g/L组。透射电子显微镜观察细胞形态。噻唑蓝比色法检测细胞增殖;实时定量聚合酶链反应、蛋白质免疫印迹法检测细胞内细胞周期蛋白D(CyclinD)、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)mRNA和蛋白相对表达量。体内实验:BALB/C小鼠于前肢背部皮下注射MuM2B细胞悬液制备移植瘤模型。建模成功后随机分为空白组和不同浓度苦参碱处理组。空白组小鼠瘤周皮下注射磷酸盐缓冲液;不同浓度苦参碱处理组小鼠瘤周皮下分别注射15、25、50、100 mg/kg苦参碱,连续7 d。末次给药后称取瘤体重量并测量其体积。多组间比较采用单因素方差分析;组间两两比较采用t检验。结果对照组细胞结构正常,分布均匀,未见或少见核固缩;不同浓度苦参碱组随浓度升高,细胞核固缩逐渐增多,细胞形态出现明显变化。与对照组比较,苦参碱0.50、1.00、2.00 g/L组细胞存活率随苦参碱浓度升高及作用时间延长,细胞存活率逐渐降低,细胞内CyclinD、Bcl-2 mRNA和蛋白相对表达量逐渐降低,Bax mRNA和蛋白相对表达量逐渐升高;同一放射剂量X线照射下,苦参碱0.50、1.00、2.00 g/L组细胞存活率逐渐降低,差异均有统计学意义(P<0.05)。与空白组比较,不同剂量苦参碱组小鼠肿瘤重量显著降低,体积显著缩小,差异有统计学意义(P<0.05)。结论苦参碱通过下调CyclinD、Bcl-2表达,上调Bax表达,促进人脉络膜黑色素瘤细胞凋亡,抑制细胞增殖,并可提高细胞放射敏感性。

Objective To investigate the effects of matrine on proliferation,apoptosis and radiotherapy sensitivity of uveal melanoma cells.Methods An animal experiment study.In vitro experiment:MuM2B cells of human choroidal melanoma were randomly divided into control group and matrine 0.25,0.50,1.00,2.00 g/L groups.The cell morphology was observed by transmission electron microscope.Cell proliferation was detected by thiazole blue colorimetry.The mRNA and relative expression levels of CyclinD D(CyclinD),B lymphoblastoma-2(Bcl-2)and Bcl2-associated X protein(Bax)were detected by real-time polymerase chain reaction and Western blot.In vivo experiment:BALB/C mice were injected with MuM2B cell suspension subcutaneously on the back of forelimb to prepare transplanted tumor model.After successful modeling,they were randomly divided into blank group and matrine treatment group with different concentrations.Mice in blank group were injected with phosphate buffer subcutaneously.Mice in different matrine treatment groups were injected with 15,25,50,100 mg/kg matrine subcutaneously,respectively,for 7 consecutive days.The tumor was weighed and its volume was measured after the last administration.Single factor analysis of variance was used to compare different groups.The t test was used for pairwise comparison between groups.Results In the control group,the cell structure was normal,the distribution was uniform,and no or rare nuclear pyknosis was seen.With the increase of matrine dosage,the nuclear pyretosis increased gradually and cell morphology changed obviously.Compared with the control group,the cell survival rate in 0.50,1.00 and 2.00 g/L groups gradually decreased with matrine concentration increasing and treatment time prolongating,the relative expression levels of CyclinD and Bcl-2 mRNA and protein gradually decreased,and the relative expression levels of Bax mRNA and protein gradually increased.Under the same radiation dose X-ray irradiation,the cell survival rate of 0.50,1.00 and 2.00 g/L groups gradually decreased,and the differences were statistically significant(P<0.05).Compared with blank group,the tumor weight and volume of mice in different doses of matrine group were significantly decreased,and the differences were statistically significant(P<0.05).Conclusion Matrine can down-regulate the expression of CyclinD and Bcl-2,up-regulate the expression of Bax,promote the apoptosis of MuM2B human melanoma cells,inhibit cell proliferation,and enhance cell radiosensitivity.