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目的观察γ线放射后FLCN缺失的肾癌细胞与高表达FLCN的肾癌细胞凋亡及自噬水平上的差别,并进一步确定γ线放射在FLCN缺失的肾癌细胞中诱导自噬的分子机制。方法本实验使用UOK257/UOK257-2及ACHN-sc/ACHN 5968两组细胞,通过Western blot、GFP-LC3及MDC荧光方法观察细胞内自噬现象,通过TUNEL方法测定细胞凋亡,通过克隆存活实验测定细胞对γ线放射的敏感性,并利用3-MA或Beclin 1 siRNA抑制自噬后,再次观察细胞内自噬、凋亡以及细胞对γ线放射敏感性的变化。结果相对于ACHN-sc/UOK257-2细胞,UOK257/ACHN 5968对γ线放射较敏感,且γ线放射可以在UOK257/ACHN 5968细胞中诱导更明显的自噬反应。应用自噬诱导剂雷帕霉素可以增强γ线放射对UOK257和ACHN 5968细胞的杀伤作用。Western blot结果表明,γ线放射通过激活MAPK信号通路并上调Beclin-1蛋白表达从而在UOK257/ACHN 5968细胞中激活自噬反应。结论 FLCN可以降低肾癌细胞对γ线放射的敏感性,无FLCN表达的UOK257肾癌细胞对γ线放射较敏感。与单独的γ线放射相比,联合应用自噬诱导剂和γ线放射对FLCN缺失或下调的肾癌细胞有更强的杀伤效果,而这可能为BHD伴发的肾癌或其它相关肿瘤的治疗提供一种更有效的治疗途径。
Objective To observe the differences of apoptosis and autophagy between FLCN-deficient renal cancer cells and FLCN-expressing renal carcinoma cells after γ-ray irradiation, and to further confirm the molecular mechanism of γ-ray emission in autophagy induced by FLCN-deficient kidney cancer cells . Methods UOK257 / UOK257-2 and ACHN-sc / ACHN 5968 cells were used in this experiment. The autophagy was observed by Western blot, GFP-LC3 and MDC fluorescence methods. The apoptosis was detected by TUNEL method. The sensitivity of the cells to γ-ray emission was measured. After autophagy was inhibited by 3-MA or Beclin 1 siRNA, the changes of autophagy, apoptosis and radiosensitivity of the cells to γ-ray were observed again. Results UOK257 / ACHN 5968 is more sensitive to gamma radiation than ACHN-sc / UOK257-2 cells and gamma radiation can induce more pronounced autophagy in UOK257 / ACHN 5968 cells. The application of rapamycin induced by autophagy can enhance the killing effect of γ-ray on UOK257 and ACHN 5968 cells. Western blot results showed that γ-ray emission activated autophagy in UOK257 / ACHN 5968 cells by activating MAPK signaling pathway and up-regulating Beclin-1 protein expression. Conclusion FLCN can reduce the sensitivity of renal cancer cells to γ-ray radiation. UOK257 cells without FLCN expression are more sensitive to γ-ray emission. Combined with autophagy-inducing agents and γ-ray irradiation, the combination of autophagy-inducing agents and γ-ray radiation has a stronger killing effect on the renal cancer cells with or without FLCN deletion, which may be associated with renal cancer or other related tumors of BHD Treatment provides a more effective treatment.