中文摘要：

背景与目的：Ca^2＋在维持细胞生物活性方面扮演着很重要的角色，其在细胞内的储存、释放和摄取主要受内质网调节，细胞内Ca^2＋浓度的稳态是维持细胞生物能量代谢、蛋白质折叠和分泌的基础条件。本研究探讨Ca^2＋在顺铂诱导SKOV3细胞内质网应激．自噬反应中的作用机制。方法：取人卵巢癌SKOV3细胞系为研究对象，按以下步骤分组：①探讨顺铂诱导内质网应激与自噬反应，用6μg／mL的顺铂处理SKOV3细胞0、6、12和24h；②了解顺铂和毒胡萝卜内酯（thapsigargin，TG）诱导内质网应激释放的Ca^2＋与自噬的关系，分别用TG和顺铂处理SKOV3细胞0、9和12h；③探究Ca^2＋对自噬的作用机制，分成对照组、顺铂组、TG组、BAPTA—AM组、顺铂联合BAPTA—AM组和TG联合BAPTA—AM组。用蛋白[质]印迹法（Western blot）检测内质网应激相关蛋白GRP78和自噬标志性蛋白LC3蛋白的表达水平；用Fluo-4钙离子荧光探针检测细胞质中的Ca^2＋浓度变化；间接免疫荧光染色后，用共聚焦显微镜检测LC3蛋白的表达情况。结果：SKOV3细胞经6μg/mL顺铂作用6h时GRP78灰度值（1．393±0．004）与其对照组（0．679±0．011）相比显著提高（t=113．2，P=0．000），在12h时LC3灰度值（0．072±0．002）与其对照组（O．038±0．000）相比显著提高（t=-25．5，P=0．000）。间接免疫荧光结果显示，顺铂（6μg／mL）组和TG（3μmol／L）组随作用时间的延长，细胞内LC3荧光斑点会逐渐增多，并伴随着细胞质Ca^2＋浓度上升。后经钙离子络合剂BAPTA．AM干预后，细胞内LC3荧光强度进一步增强。Westren blot结果显示，顺铂组LC3灰度值（0．039±0．000）小于顺铂联合BAPTA—AM组（0．071±0．001），TG组（0．035±0．001）小于TG联合BAPTA-AM组（0．065±0．001），差异有统计学意义（P=0．000）。结论：顺铂诱导SKOV3细胞内质网应激和自噬的发生?

英文摘要：

Background and purpose： Ca^2＋ plays a very important role in the maintenance of cell biological functions. The storage, release and uptake capacity of Ca^2＋ is controlled by endoplasmic reticulum （ER）. Ca^2＋ homeostasis is essential for cellular energy metabolism and proper protein folding. This study aimed to investigate the effect of cytoplasmic Ca^2＋ on cisplatin induced ER stress-mediated autophagy in ovarian carcinoma SKOV3 and its underlying mechanism. Methods： The ovarian cancer SKOV3 was used as a study object. The experiment consisted of three parts： ① To explore the possible relationship between cisplatin-induced ER stress and autophagy, SKOV3 cells were treated with cisplatin for 0, 6, 12 and 24 h, respectively; ②To explore the possible relationship between ER stress induced Ca^2＋ efflux and autophagy, SKOV3 cells were treated with cisplatin for 0, 9 and 12 h, respectively, and TG was used as a positive control;③ To explore the effects of blocking calcium efflux on autophagy, SKOV3 cells were divided into control group, cisplatin group, TG group, BAPTA-AM group, cisplatin combined with BAPTA-AM group and TG combined with BAPTA-AM group. Western blot was used to detect the protein levels of GRP78 and LC3. Fluo-4 calcium fluorescent probe was used to examine cytoplasmic Ca^2＋ levels. Confocal microscopy was used to detect LC3 level by immunoflurescence staining. Results： Compared to control group （0.679±0.011）, GRP78 was significantly accumulated at 6, 12 and 24 h after cisplatin treatment and reached the maximum value at 6 h （1.393±0.004, P=0.000）. Similarly, compared to control group （0.038±0.000）, LC3 puncta were clearly seen after cisplatin treatment and reached the maximum value at 12 h （0.072±0.002, P=0.000）. Using confocal microscopy, we found that cisplatin and TG increased LC3 punctate accumulation and cytoplasmic Ca^2＋ levels in a time-dependent manner. Immunofluorescent method showed that treatment with cisplatin combined with BAPTA-AM or TG