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目的:通过对自噬基因Beclin1的表达调节来观察细胞自噬对紫杉醇诱导人宫颈癌CaSki细胞株死亡的影响,并探讨在该过程中自噬与凋亡之间的相互作用及关系。方法:利用脂质体法将Bec-lin1的过表达质粒pcDNA3.1-Beclin1和RNA干扰质粒pSUPER-Beclin1分别体外转染CaSki细胞并筛选稳定表达株后,电镜下观察细胞内自噬囊泡的形成,Western印迹检测转染前后Beclin1与LC3蛋白的表达变化。上述细胞株经紫杉醇作用后MTT法检测细胞增殖情况的改变,流式细胞仪检测自噬细胞与凋亡细胞的百分率。结果:在Beclin1基因过度表达的CaSki细胞株中,电镜观察到细胞内存在大量的自噬囊泡;Beclin1与LC3蛋白的表达在pcDNA3.1-Beclin1转染细胞株中被上调。MTT法检测提示Beclin1过表达细胞株存活细胞明显少于未转染对照细胞株。流式细胞仪检测提示经紫杉醇作用后与空白对照组比较,Beclin1过表达组自噬细胞与凋亡细胞百分率均有明显增加,其中凋亡增加尤其明显;而Beclin1干扰组自噬细胞与凋亡细胞百分率均有所减少。结论:在紫杉醇对宫颈癌CaSki细胞株的杀伤过程中细胞自噬与凋亡均发挥了作用,在Beclin1基因过度表达的CaSki细胞株中可通过增加紫杉醇诱导的细胞凋亡来增强药物对肿瘤细胞的杀伤作用。
OBJECTIVE: To observe the effect of autophagy on autophagy gene Beclin1 expression in paclitaxel-induced human cervical cancer CaSki cell line and to explore the relationship between autophagy and apoptosis in this process. METHODS: Bec-lin1 overexpression plasmid pcDNA3.1-Beclin1 and RNA interference plasmid pSUPER-Beclin1 were transfected into CaSki cells by lipofectamine 2000 respectively and the stable expression strains were screened. The expression of autophagic vesicles The expression of Beclin1 and LC3 protein was detected by Western blotting. The above cell lines were treated with paclitaxel and the cell proliferation was detected by MTT assay. The percentage of autophagic cells and apoptotic cells was detected by flow cytometry. Results: In the CaSki cell line overexpressing Beclin1, a large number of autophagic vesicles were observed in the cells by electron microscopy. The expression of Beclin1 and LC3 proteins was up-regulated in pcDNA3.1-Beclin1 transfected cell lines. MTT assay showed that Beclin1 overexpression cell line survival cells were significantly less than the untransfected control cell line. Flow cytometry showed that after paclitaxel treatment compared with the control group, Beclin1 over-expression of the percentage of apoptotic cells and autophagy cells were significantly increased, especially in increased apoptosis; Beclin1 interference group with autophagy and apoptosis The percentage of cells decreased. CONCLUSION: Paclitaxel plays a role in the autophagy and apoptosis of cervical cancer cell line CaSki during the killing process. Paclitaxel-induced apoptosis in CaSki cell line with over-expression of Beclin1 gene can enhance the effect of drugs on tumor cells The killing effect.