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目的:研究不同电离辐射方式对卵巢癌耐药细胞株SKVCR自噬性细胞死亡的影响,并探讨其相关机制。方法:实验分为假照组、分割照射组(2Gy·d-1×5)及单次照射组(10Gy·d-1×1)。采用MTT法检测各组细胞对长春新碱(VCR)、依托泊苷(VP-16)及顺铂(DDP)的药物敏感性,MDC染色及流式细胞术检测自噬发生率的变化,实时荧光定量PCR方法检测自噬特异基因MAPLC3B和Akt1mRNA水平,Western blotting法检测自噬相关蛋白MAPLC3B表达和蛋白激酶B(PKB,Akt1)/哺乳动物雷帕霉素靶蛋白(mTOR)及其下游基因P70S6K、磷酸化AKT1/mTOR/P70S6K表达的变化。结果:与假照组比较,电离辐射使SKVCR细胞对VCR、VP-16的药物敏感性提高,分割照射组更明显(P<0.05)。与假照组比较,电离辐射使细胞自噬发生率升高,尤其以分割照射组升高更明显(P<0.05);与假照组比较,照射后MAPLC3B mRNA升高、Akt1mRNA下降(P<0.05);照射后MAPLC3B蛋白表达升高,Akt1、mTOR、p-mTOR、P70S6K、p-P70S6K蛋白表达均不同程度下降,分割照射组较单次照射组下降更明显(P<0.05)。结论:不同的电离辐射作用方式可以引起卵巢癌细胞发生自噬性死亡,其机制主要涉及Akt1/mTOR/S6K通路。
Objective: To study the effect of different ionizing radiation on the death of ovarian cancer cell line SKVCR and to explore the related mechanisms. Methods: The experiment was divided into sham group, split irradiation group (2Gy · d-1 × 5) and single irradiation group (10Gy · d-1 × 1). The chemosensitivity of vincristine (VCR), etoposide (VP-16) and cisplatin (DDP) was detected by MTT assay. The changes of autophagy were detected by MDC staining and flow cytometry. The levels of MAPLC3B and Akt1mRNA were detected by real-time fluorescence quantitative PCR. The expression of MAPLC3B and PKB (Akt1) / mammalian target of rapamycin (mTOR) and its downstream gene P70S6K , Phosphorylation of AKT1 / mTOR / P70S6K expression changes. Results: Compared with the sham group, the sensitivity of SKVCR cells to VCR and VP-16 was enhanced by ionizing radiation and the irradiation was more obvious (P <0.05). Compared with the sham group, the incidence of autophagy increased, especially in the split group (P <0.05). Compared with sham group, MAPLC3B mRNA and Akt1 mRNA decreased (P < 0.05). After irradiation, the expression of MAPLC3B protein increased. The expression of Akt1, mTOR, p-mTOR, P70S6K and p-P70S6K all decreased to different extents, and the fractionated irradiation group was more obvious than the single irradiation group (P <0.05). Conclusion: Different ionizing radiation modes can cause autophagic cell death in ovarian cancer cells. The mechanism is mainly related to the Akt1 / mTOR / S6K pathway.