双氢青蒿素对人骨肉瘤细胞株143B增殖和凋亡的作用

Role of dihydroartemisinin(DHA) in proliferation and apoptosis of human osteosarcoma 143B cells

目的观察双氢青蒿素(dihydroarteminin,DHA)对人骨肉瘤细胞143B的增殖和凋亡的影响以及可能的机制。方法体外培养人骨肉瘤细胞株143B;MTT比色法和克隆形成实验检测不同浓度DHA对骨肉瘤细胞存活与克隆形成能力的影响。Hoechst 33258染色法观察细胞凋亡的形态变化。构建β-Catenin荧光素酶报告基因(β-Catenin-Luc,p-Top)检测DHA作用143B后细胞内β-Catenin活性变化。不同浓度的DHA作用后,Western blot检测与细胞增殖(如PC-NA、Cyclin D1、c-Myc)、凋亡(如Bad、Bcl-2、Caspase-3)密切相关的标志物蛋白质表达变化。结果 DHA作用于143B细胞24 h后,MTT结果显示143B细胞的增殖活性受到明显抑制,且其克隆形成能力减弱(P<0.05),在DHA浓度达35μmol.L-1时,抑制效率最明显。Western blot结果显示PC-NA表达下调;而促凋亡蛋白Bad和Caspase-3表达上调,Bcl-2蛋白表达明显减弱。结论双氢青蒿素具有较明显的抑制人骨肉瘤细胞的增殖且促进其凋亡的作用,可能通过下调细胞增殖相关蛋白PCNA、Bcl-2和上调促凋亡蛋白Bad和Caspase-3,启动凋亡程序,致143B细胞发生凋亡。

Aim To observe the effect of dihydroartemisinin（DHA） on proliferation and apoptosis of human osteosarcoma 143B cells and the possible mechanism.Methods Human osteosarcoma cell line 143B was cultured in vitro;MTT [3-（4,5） the-dimethylthiahiazo（-z-y1）-3,5-di-phenytetrazoliumromide,MTT] colorimetric assay and clone formation assay were applied to detect the role of DHA in the survival and growth status of human osteosarcoma cells.Hoechst 33258 cell staining was used for apoptosis detection.The luciferase reporter gene,β-Catenin-Luc（p-Top） was constructed to detect β-Catenin activity after DHA＇s role in 143B cells.The marker protein expression closely related to tumor cell proliferation,such as PCNA,Cyclin D1,C-Myc,and apoptosis,such as Bad,Bcl-2,Caspase-3,was detected by Western blot.Results 24 hours after DHA played role in 142B,MTT results showed that DHA could inhibit the proliferation of the 143B cells,and colony formation diminished（P0.05）,the inhibition efficiency increased significantly at a concentration of 35 μmol·L-1DHA.143B cells was significantly enhanced（P0.05）,and the PCNA expression was lowered,and cell proliferation was inhibited,pro-apoptotic protein Bad and caspase-3 expression was significantly enhanced,while Bcl-2 protein expression decreased significantly.Conclusions Dihydroartemisinin significantly inhibits the proliferation of human osteosarcoma 143B cells and promote apoptosis,which may be associated with the downregulation of cell proliferation-associated proteins,the PCNA and Bcl-2,and the upregulation of pro-apoptotic protein,Bad and Caspase-3.