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目的:检测小鼠黑色素瘤细胞株B16促红细胞生成素受体(erythropoietin receptor,EPOR)的表达,探讨促红细胞生成素(erythropoietin,EPO)对达卡巴嗪和顺铂抗B16细胞活性的影响及与Bcl-2家族蛋白表达的关系。方法:采用RT-PCR和细胞免疫化学染色法检测B16细胞EPOR mRNA和蛋白的表达;MTT法和克隆形成试验检测EPO与达卡巴嗪或顺铂联合应用时对B16细胞增殖的影响;实时荧光定量RT-PCR和蛋白质印迹法检测EPO对B16细胞Bcl-2、Bcl-xL、Bax mRNA和蛋白表达的影响。结果:B16细胞表达EPOR mRNA和蛋白。EPO能有效减少达卡巴嗪诱导B16细胞的凋亡(P<0.05),但未能显著影响顺铂对B16细胞的作用,P>0.05。B16细胞经EPO处理后,其Bcl-2、Bcl-xL mRNA和蛋白表达水平明显增加(P<0.05),Bax mRNA和蛋白表达水平未见明显变化,P>0.05。结论:B16细胞表达EPOR,EPO可调节化疗药对B16细胞的抗肿瘤活性,且该作用与化疗药的类型有关,其作用机制可能与EPO影响Bcl-2家族蛋白表达有关。
OBJECTIVE: To detect the expression of erythropoietin receptor (EPOR) in mouse melanoma cell line B16 and investigate the effect of erythropoietin (EPO) on the anti-B16 cell activity of dacarbazine and cisplatin Bcl-2 family protein expression. Methods: The expression of EPOR mRNA and protein in B16 cells was detected by RT-PCR and immunocytochemistry. The effects of EPO combined with dacarbazine or cisplatin on the proliferation of B16 cells were detected by MTT assay and colony formation assay. The real-time fluorescence quantitative The effects of EPO on the expression of Bcl-2, Bcl-xL, Bax mRNA and protein in B16 cells were detected by RT-PCR and Western blot. Results: B16 cells expressed EPOR mRNA and protein. EPO can effectively reduce the apoptosis of B16 cells induced by dacarbazine (P <0.05), but did not significantly affect the effect of cisplatin on B16 cells, P> 0.05. After EPO treatment, Bcl-2 and Bcl-xL mRNA and protein levels were significantly increased in B16 cells (P <0.05), and Bax mRNA and protein expression levels were not significantly changed (P> 0.05). Conclusion: B16 cells express EPOR, EPO can regulate the anti-tumor activity of chemotherapeutic drugs on B16 cells, and the effect is related to the type of chemotherapeutic drugs. The mechanism may be related to the effect of EPO on Bcl-2 family protein expression.