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目的初步探讨TFAR19协同米非司酮(MIF)对前列腺癌PC-3M细胞凋亡的影响。方法构建TFAR19真核表达载体,用脂质体介导的方法转染PC-3M细胞。MTT法检测5、10、20、50和100μmol·L-1MIF作用于前列腺癌PC-3M细胞24~96h的吸光度(A)值。在转染TFAR19的细胞中加入20mol·L-1MIF培养24、48h,MTT比色法检测细胞增殖,原位末端标记(TUNEL)法检测细胞凋亡率,透射电镜进一步观察细胞超微结构的改变。结果构建了PCI-neo-TFAR19真核表达载体并在转染的PC-3M细胞中得到了瞬时表达。MTT实验表明,与对照组相比,5、10μmol·L-1MIF组的A值差异无统计学意义(P>0.05),20、50和100μmol·L-1MIF组的A值差异有统计学意义(P<0.01),MIF对前列腺癌PC-3M细胞的抑制作用呈时间、剂量依赖性;转染PCI-neo-TFAR19并加入20 mol·L-1MIF后,与对照组及单独应用MIF组相比,细胞生长明显受到抑制(P<0.01),细胞凋亡率明显增加(P<0.01),透射电镜观察到典型的细胞凋亡特征(细胞体积缩小,核皱缩、碎裂,染色质呈块状边集等)。结论TFAR19蛋白能够协同米非司酮促进前列腺癌PC-3M细胞凋亡,有望成为前列腺癌的辅助治疗药物。
Objective To investigate the effect of TFAR19 in combination with mifepristone (MIF) on the apoptosis of prostate cancer PC-3M cells. Methods TFAR19 eukaryotic expression vector was constructed and transfected into PC-3M cells by liposome-mediated method. The MTT assay was used to detect the absorbance (A) value of 5, 10, 20, 50 and 100μmol·L-1MIF in prostate cancer PC-3M cells 24-96h. The cells transfected with TFAR19 were cultured in 20mol·L-1MIF for 24,48 h. The cell proliferation was detected by MTT colorimetric assay. The apoptosis rate was detected by TUNEL assay. The ultrastructural changes were observed by transmission electron microscopy . Results The eukaryotic expression vector PCI-neo-TFAR19 was constructed and transiently expressed in transfected PC-3M cells. The results of MTT assay showed that there was no significant difference in A value between 5 and 10 μmol·L-1 MIF group (P> 0.05), but the A value of 20, 50 and 100 μmol·L-1 MIF group was statistically significant (P <0.01). The inhibitory effect of MIF on prostate cancer PC-3M cells was time-and dose-dependent. After transfection with PCI-neo-TFAR19 and 20 mol·L-1MIF, (P <0.01), and the apoptosis rate was significantly increased (P <0.01). The typical apoptotic characteristics (such as cell volume shrinkage, nuclear shrinkage, fragmentation, chromatin were observed by transmission electron microscope Block edge set, etc.). Conclusion TFAR19 protein can cooperate with mifepristone to promote the apoptosis of prostate cancer PC-3M cells, and is expected to be adjuvant therapy for prostate cancer.