CP节律性化疗对人脐静脉内皮细胞及其中自噬相关蛋白Beclin1、LC3的影响

Effect of CP metronomic chemotherapy on HUVEC cells and autophagy-related protein Beclin1 and LC3

目的研究持续低剂量磷酰胺氮芥(phosphoamide,PM)联合泼尼松龙(CP节律性化疗方案)对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)自噬相关蛋白Beclin1和LC3表达水平的影响,探讨节律性化疗增加HUVEC对化疗药物敏感性的机制。方法实验分DMSO对照组、磷酰胺氮芥(PM)组、泼尼松龙组、磷酰胺氮芥+泼尼松龙组(CP组),每组重复3次。采用不同的药物处理HUVEC,用CCK8法检测细胞增殖,流式细胞术检测细胞凋亡,免疫印迹法和反转录PCR检测自噬相关蛋白的表达水平。结果 (1)与DMSO对照组相比,随着时间的延长,PM、泼尼松龙及CP组均使HUVEC的增殖抑制率明显升高(P均<0.001),并且在相同作用时间,不同组的药物对细胞的增殖抑制率也明显不同,泼尼松龙组对细胞的抑制率最低,而CP组对细胞的增殖抑制率(72 h,25.1%±0.7%)最高,PM组(72 h,20.4%±0.7%)次之(P均<0.01);(2)用药处理48 h后,与DMSO对照组相比,泼尼松龙组、PM组、CP组使HUVEC细胞的凋亡率依次增高(P均<0.001);(3)用药处理48 h后,与DMSO对照组相比,泼尼松龙组、PM组、CP组的Beclin1、LC3的蛋白表达和mRNA表达水平均依次下降(P均<0.001)。结论人脐静脉内皮细胞HUVEC经CP节律性化疗方案处理后,可以显著降低HUVEC细胞的增殖水平,促进HUVEC细胞的凋亡,并显著降低自噬相关蛋白Beclin1和LC3的表达水平。

Objective To investigate the effect of CP metronomic chemotherapy on HUVEC cells and autophagy-related pro-tein, Beclin1 and LC3, expression levels, and to explore the mechanism of CP program increasing sensitivity of HUVEC cells to che-motherapeutic drug.Methods Subjects were divided into the DMSO control group, and the phosphoramide mustard （PM） group, the prednisolone group, the phosphoramide mustard and prednisolone group （the CP group）.HUVEC cells were treated with different drugs.CCK8 method was used to detect cell proliferation, flow cytometry to detect the cell apoptosis, Western blot and RT-PCR to de-tect autophagy-related protein levels.Results CCK8 method showed that compared with DMSO control group, in the PM, the prednis-olone and the CP groups, HUVEC cell proliferation inhibition rates increased significantly with the extension of time （P〈0.001）.And at the same time, the different drug groups were significantly different in cell proliferation inhibition rate, the prednisone group was the weakest, the CP group（72 h,25.1%±0.7%）was the strongest, and the PM group （72 h,20.4%±0.7%） was intermediate（P〈0.01）.Flow cytometry showed that after 48 hours, compared with the DMSO control group, the prednisolone, the PM and the CP groups made HUVEC cell apoptosis rate increased in turn （P〈0.001）.Western blot and reverse transcription PCR showed that after 48 hours, compared with the DMSO control group, Beclin1 and LC3 protein and mRNA expression decreased in turn in the predniso-lone group, the PM group and the CP group（P〈0.001）.Conclusions HUVEC cells treated with CP metronomic chemotherapy can significantly reduce the level of HUVEC cell proliferation, promote HUVEC cell apoptosis, and significantly reduce autophagy-related protein expression levels of Beclin1 and LC3.