顺铂与卡铂对骨肉瘤细胞株作用的分子机制

Molecular mechanism of cisplatin and carboplatin on osteosarcoma cell line

目的探讨顺铂与卡铂对骨肉瘤细胞(MG-63)作用的分子机制。方法骨肉瘤细胞混悬液与不同浓度顺铂和卡铂分别作用24、48、72 h后,用倒置显微镜观察细胞生长情况。用噻唑蓝法测定每孔的吸光度值,制作生长曲线,并求出顺铂和卡铂的半抑制浓度(IC_(50))。用IC_(50)顺铂、卡铂作用于骨肉瘤细胞24、48、72 h后用流式细胞仪进行细胞周期分析。免疫印迹技术观察细胞的增殖细胞核抗原(PCNA)、Bcl-2和Bax的表达。结果相同浓度顺铂和卡铂作用的骨肉瘤细胞死亡数量随时间增加而增加。不同浓度顺铂和卡铂对骨肉瘤细胞分别作用24、48、72 h后,对细胞的生长均起到抑制作用,相同时间内,药物浓度越高,生长抑制作用越明显(P<0.05)。骨肉瘤细胞与IC_(50)顺铂和卡铂作用24、48、72 h后,在细胞各期均未见明显凋亡峰,但随着时间增加,G_1、S、G_2期所占比例逐渐下降;顺铂和卡铂作用后不同时间点凋亡细胞所占比例比较差异均有统计学意义(P<0.05)。与对照组比较,顺铂组和卡铂组PCNA表达下降,Bax表达增加,Bcl-2表达略减少。结论顺铂和卡铂对肿瘤细胞生长各期均有抑制作用,对于骨肉瘤细胞来说,卡铂的浓度约需25倍于顺铂才能产生相似的生长抑制效应。顺铂、卡铂与骨肉瘤细胞作用后存在凋亡现象,但是未能形成凋亡峰,这可能与铂类化合物能够对处于任何生长周期的细胞均能产生杀伤作用有关。

Objective To explore the molecular mechanism of cisplatin and carboplatin on osteosarcoma cells（ MG-63）. Methods The osteosarcoma cell suspension was interacted with different concentrations of cisplatin and carboplatin for24,48,72 h; then the cell growth was observed with inverted microscope. The absorbance value of each hole was determined by methyl thiazolyl tetrazolium（ MTT） method,and the growth curve based on above results was drawed,and the half maximal inhibitory concentration（ IC_（50）） of cisplatin and carboplatin was calculated. After IC_（50） concentrations of cisplatin,carboplatin interacted with osteosarcoma cells for 24,48,72 h,the cell cycle was analysed by flow cytometry. The expression of proliferating cell nuclear antigen（ PCNA）,Bcl-2 and Bax was observed by western blot technique. Results The number of osteosarcoma cell death increased with the increase of time of the same concentration of cisplatin and carboplatin interacted with osteosarcoma cell. The cell growth inhibition was observed after different concentration of cisplatin and carboplatin interacted with osteosarcoma cell for 24,48,72 h. At the same time point,the greater the concentration of the drug,the more obvious the growth inhibition（ P 0. 05）. There was no obvious peaks of apoptosis in the each cell cycle phases after IC_（50） cisplatin and carboplatin interacted with osteosarcoma cell for 24,48,72 h; but with the increase of time,the proportion of G1,S,G2 stage cell gradually decreased; there was statistic difference of the proportion of apoptotic cells at different time after cisplatin or carboplatin interacted with osteosarcoma cell（ P 0. 05）. The expression of PCNA in cisplatin and carboplatin group decreased compared with control group; the expression of Bax increased compared with control group; the expression of Bcl-2 was slightly reduced. Conclusion Cisplatin and carboplatin have inhibition role on the growth of the each period of tumor cells. For osteosarcoma cells,the concentration of carboplatin is about 25 times of cisplatin can produce similar growth inhibition effect. There are apoptosis after cisplatin and carboplatin interacted with the osteosarcoma cell,but failed to form a peak of apoptosis,which may be associated with platinum-based compounds can kill each period of cells in the cell cycle.