伊立替康对RAW264．7巨噬细胞增殖和细胞凋亡的影响

Effects of Irinotecan on proliferation and apoptosis of RAW 264.7 macrophages

目的分析抗肿瘤药伊立替康(CPT-11)对RAW 264.7巨噬细胞增殖及细胞凋亡的影响,并探讨其作用机制。方法WST-1法检测CPT-11对RAW 264.7细胞增殖的作用,倒置显微镜观察CPT-11对RAW 264.7细胞形态的影响,Hoechst33342染细胞核观察RAW 264.7细胞的核形态,流式细胞术检测亚二倍体(凋亡峰)的变化,免疫印迹法检测Caspase-3活化、PARP及细胞周期相关蛋白的变化。结果 CPT-11能明显抑制RAW 264.7细胞增殖,并呈时间和剂量依赖性。在CPT-11作用下RAW 264.7细胞体积增大,细胞核增大,部分细胞发生核碎裂(细胞凋亡),并且原本贴壁生长、具有伪足的细胞变圆脱落。流式细胞术结果也显示,CPT-11可诱导RAW 264.7细胞产生亚二倍体峰(凋亡细胞),且细胞凋亡率呈浓度依赖性上升。同时,处于G2/M期细胞也明显增多,且呈浓度依赖性。免疫印迹分析显示,CPT-11可活化Caspase-3,使PARP水平下降,同时上调细胞周期蛋白Cyclin B1,下调Cyclin D1的表达。结论伊立替康通过诱导细胞周期阻滞而抑制RAW 264.7细胞增殖,通过激活Caspase-3通路诱导细胞凋亡。

This study aimed to explore the effect of anticancer agent Irinotecan （CPT-11） on cell proliferation and apoptosis in RAW 264.7 cells and the underlying mechanism. Cell proliferation was measured by WST-1 assay; cell morphology was observed with an inverted microscope. Nucleus morphology was revealed by Hoechst 33342 staining and the percentage of apoptotic cells was calculated. The ratios of cells at each cell cycle stage, including sub-G0/G1, were analyzed by flow cytometry. Immunoblotting was recruited to evaluate the levels of cleaved caspase-3, PARP and cell cycle related proteins. The results showed that CPT-11 inhibited the proliferation of RAW 264.7 cells in a time- and dose-dependent manner. Under the action of CFF-11, RAW 264.7 cells and their nuclei became bigger, the pseudopodium protrusions disappeared and the cells became round compared with control groups; furthermore, CPT-11 induced nuclear fragmentation （apoptosis） of RAW 264.7 cells in a dose-dependent manner. The ratios of cells at hypodiploid peak （apoptotic peak） and GgM phase were increased with the increase of CPT-11 doses, indicating the induction of cell apoptosis and cell cycle arrest. Moreover, CPT-11 also resulted in activation of caspase-3, decreased PARP, as well as increased levels of cyclin B1, but decreased levels of cyclin D1. Our data demonstrated that CPT-11 can inhibit proliferation of RAW 264.7 ceils by arresting cell cycle and induce RAW 264.7 cells apoptosis through the caspase-3 pathway.