苦参碱对人横纹肌肉瘤RD细胞增殖及凋亡的影响及其作用机制

Effects and mechanisms of matrine on proliferation and apoptosis of human rhabdomyosarcoma RD cells line

目的观察苦参碱对体外人横纹肌肉瘤（RMS）RD细胞株增殖和凋亡的影响，研究苦参碱对人RMSRD细胞株中β-catenin与原癌基因Bcl-2及凋亡基因Caspase-3的影响及其表达关系，探索Wnt／β-catenin通路在RMS发生过程中的作用机制。方法应用不同质量浓度苦参碱（0．5、1．0、1．5、2．0g／L）分别作用人RMSRD细胞株48h；采用四甲基偶氮唑蓝（MTT）比色法检测不同水平苦参碱对RD细胞的增殖抑制率；流式细胞仪检测细胞凋亡率；Western blot法检测β-catenin、Bcl-2及Caspase-3蛋白的表达。结果MTT检测对照组及各实验组增殖抑制率分别为（13．70±0．25）％、（33．16±0．11）％、（42．96±0．90）％、（56．26±0．79）％和（67．89±0．63）％；流式细胞仪检测对照组及各实验组凋亡率分别为（5．49±0．96）％、（17．23±5．03）％、（25．84±4．17）％、（36．08±3．68）％、（47．79±4．82）％；Western blot结果显示对照组β-catenin和Bcl．2蛋白表达最高，而Caspase-3表达量最低，各浓度组β-catenin和Bcl-2的表达明显降低，而Caspase-3的表达明显增高，且表现明显的质量浓度依赖性。以上检测各浓度组与对照组比较差异均有统计学意义（P均〈0．05）。结论苦参碱能明显抑制RMSRD细胞的增殖，并诱导其凋亡；苦参碱可下调Wnt/β-catenin通路中关键分子β-catenin的表达，降低Bcl-2的表达，上调Caspase-3表达；Wnt/β-catenin信号通路在苦参碱诱导的RD细胞凋亡过程中发挥重要作用，其下游调控蛋白Bcl-2及Caspase-3共同参与了这一过程的调控。

Objective To investigate the effects of matrine on proliferation and apoptosis of human rhabdomyosarcoma（RMS） RD cells line in vitro, to study the regulatory mechanism of Wnt.β -catenin pathway -influenced apoptosis of RD cells line by detecting the expressions of β- catenin protein, Bcl - 2 protein and caspase - 3 protein, and to explore Wnt β - catenin mechanism during the process of RMS. Methods The human RMS RD cells line was treated with matrine of different concentrations （0.5,1.0,1.5,2.0 g/L） for 48 hours respectively, and the proliferation inhibition rates of different concentrations of matrine on RD cells were detected by methyl thiazolyl tetrazolium assay, while the apoptosis rates by flow cytometry （FCM） and the expressions of β -catenin, Bcl -2 and caspase -3 by Western blot. Results The proliferation inhibition rates between control group and different concentrations of matrine groups were（13.70 ±0.25）% ,（33.16 ±0.11）% ,（42.96 ±0.90）%, （56.26 ±0.79）% and （67.89 ±0.63）%, respectively. The apoptosis rates were （5.49 ± 0.96 ） %, （ 17.23 ± 5.03 ） %, （ 25.84 ± 4.17 ） %, （ 36.08 ± 3.68 ） % and （47.79 ± 4.82） %, respectively. The highest expression of β- catenin and Bcl -2 proteins and the minimum amount of caspase - 3 protein were found in the control group. After intervention of matrine, the expressions of β - catenin and Bcl - 2 reduced while the amount of caspase - 3 rose significantly, which was concentration - dependent obviously. Differences were found between every concentration of matrine group with control group according to statistics （ all P 〈 0.05 ）. Conclusions Matrine can inhibit proliferation and induce apoptosis of RD cells. Matrine can down - regulate the expression of β - catenin and Bcl - 2 proteins in RD cells, while the amount of caspase - 3 protein rises. Wnt/β - catenin signal pathway plays an important role in the apoptosis of RD cell induced by matrine, and its downstream proteins Bcl -2 and caspase -3 are also involved in the regulation of this process.