【摘要】：目的分析抗腫瘤藥伊立替康(CPT-11)對RAW 264.7巨噬細(xì)胞增殖及細(xì)胞凋亡的影響,并探討其作用機(jī)制。方法WST-1法檢測CPT-11對RAW 264.7細(xì)胞增殖的作用,倒置顯微鏡觀察CPT-11對RAW 264.7細(xì)胞形態(tài)的影響,Hoechst33342染細(xì)胞核觀察RAW 264.7細(xì)胞的核形態(tài),流式細(xì)胞術(shù)檢測亞二倍體(凋亡峰)的變化,免疫印跡法檢測Caspase-3活化、PARP及細(xì)胞周期相關(guān)蛋白的變化。結(jié)果 CPT-11能明顯抑制RAW 264.7細(xì)胞增殖,并呈時間和劑量依賴性。在CPT-11作用下RAW 264.7細(xì)胞體積增大,細(xì)胞核增大,部分細(xì)胞發(fā)生核碎裂(細(xì)胞凋亡),并且原本貼壁生長、具有偽足的細(xì)胞變圓脫落。流式細(xì)胞術(shù)結(jié)果也顯示,CPT-11可誘導(dǎo)RAW 264.7細(xì)胞產(chǎn)生亞二倍體峰(凋亡細(xì)胞),且細(xì)胞凋亡率呈濃度依賴性上升。同時,處于G2/M期細(xì)胞也明顯增多,且呈濃度依賴性。免疫印跡分析顯示,CPT-11可活化Caspase-3,使PARP水平下降,同時上調(diào)細(xì)胞周期蛋白Cyclin B1,下調(diào)Cyclin D1的表達(dá)。結(jié)論伊立替康通過誘導(dǎo)細(xì)胞周期阻滯而抑制RAW 264.7細(xì)胞增殖,通過激活Caspase-3通路誘導(dǎo)細(xì)胞凋亡。
[Abstract]:Objective to analyze the effect of CPT-11 on the proliferation and apoptosis of RAW 264.7 macrophages and to explore its mechanism. Methods the effect of CPT-11 on the proliferation of RAW 264.7 cells was detected by WST-1 assay, the effect of CPT-11 on the morphology of RAW 264.7 cells was observed by inverted microscope, the nuclear morphology of RAW 264.7 cells was observed by Hoechst33342 staining, the changes of subdiploid (apoptosis peak) were detected by flow cytometry, and the activation of Caspase-3, PARP and cell cycle related proteins were detected by immunoblotting. Results CPT-11 could significantly inhibit the proliferation of RAW 264.7 cells in a time-and dose-dependent manner. Under the action of CPT-11, the volume of RAW 264.7 cells increased, the nucleus increased, some cells broke into nucleus (apoptosis), and the cells with pseudopod became round and exfoliated. The results of flow cytometry also showed that CPT-11 could induce subdiploid peak (apoptotic cells) of RAW 264.7 cells, and the apoptosis rate increased in a concentration-dependent manner. At the same time, the number of cells in G _ 2 鈮，

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