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目的观察吉西他滨(gemcitabine,GEM)处理膀胱癌T24细胞后引起凋亡的同时是否诱导T24细胞自噬的产生,并初步探究其自噬产生的机制,以及自噬与凋亡之间的关系。方法体外培养T24细胞,应用CCK-8法测定细胞抑制率;Western blot检测凋亡相关蛋白Caspase-3、PARP的活化,自噬标志蛋白LC3-Ⅱ、底物蛋白P62和自噬潮,以及JNK通路相关蛋白Jnk1、P-Jnk、Bcl-2的表达情况;并通过透射电镜观察细胞超微结构自噬体的变化;Annexin V-PI双流式细胞术检测抑制自噬后细胞凋亡率的变化情况。结果 CCK-8检测结果显示吉西他滨能有效抑制膀胱癌T24细胞增殖;1.0μg/mL吉西他滨处理T24细胞4 h即可发生明显的自噬,透射电镜观察可见自噬体小体的形成;Western blot检测结果显示吉西他滨作用T24细胞后cleaved-Caspase-3、PARP和LC3-Ⅱ表达增高,P62表达降低;P-Jnk出现活化,Bcl-2发生降解;而抑制自噬后能显著增强吉西他滨诱导的膀胱癌T24细胞的凋亡。结论吉西他滨介导膀胱癌T24细胞凋亡的同时又诱导保护性自噬,JNK信号通路参与调控其自噬的发生,抑制自噬可以明显增强膀胱癌T24细胞对吉西他滨的化疗敏感性。
Objective To investigate whether gemcitabine (GEM) can induce apoptosis of bladder cancer T24 cells and induce autophagy in T24 cells, and to explore the mechanism of autophagy and the relationship between autophagy and apoptosis. Methods T24 cells were cultured in vitro and the cell inhibitory rate was determined by CCK-8 assay. The activation of caspase-3, PARP, LC3-Ⅱ, P62 and autophagy were detected by Western blot and JNK The expression of Jnk1, P-Jnk and Bcl-2 were detected by transmission electron microscopy. The changes of autophagy were observed by Annexin V-PI double flow cytometry. Happening. Results The results of CCK-8 showed that gemcitabine could effectively inhibit the proliferation of bladder cancer T24 cells. Autophagy was observed in T24 cells treated with 1.0μg / mL gemcitabine for 4 hours. The formation of autophagosomes was observed by transmission electron microscope. The results showed that the expression of cleaved-Caspase-3, PARP and LC3-II increased after gemcitabine treatment and the expression of P62 was decreased. P-Jnk was activated and Bcl-2 was degraded. Gestamil-induced bladder cancer T24 cell apoptosis. Conclusion Gemcitabine can induce the apoptosis of bladder cancer T24 cells while inducing protective autophagy. JNK signaling pathway is involved in the regulation of autophagy. Inhibition of autophagy can significantly enhance chemosensitivity of bladder cancer T24 cells to gemcitabine.