氯化钴对人卵巢癌SKOV3细胞顺铂敏感性的影响

目的：观察氯化钴（CoCl₂）作用于人卵巢癌SKOV3细胞株后对SKOV3细胞顺铂敏感性的影响，并阐述其可能机制。方法：体外培养SKOV3细胞株，随机分为对照组、CoCl₂组、顺铂（DDP）组和CoCl₂联用DDP （联合）组，CoCl₂组细胞以200 μmol·L^-1 CoCl₂作用4 h后换普通细胞培养基培养20 h，DDP组细胞以含10 mg·L^-1 DDP的细胞培养基培养24 h，联合组细胞以200 μmol·L^-1 CoCl₂作用4 h后更换含10 mg·L^-1 DDP的细胞培养基继续培养20 h。MTT法检测各组细胞存活率，免疫荧光法检测各组细胞中低氧诱导因子1α（HIF-1α）和诱导型一氧化氮合酶（iNOS）阳性表达强度，Rhod 2-AM荧光探针检测各组细胞线粒体钙离子（Ca²⁺）水平，免疫蛋白印迹（Western blotting）法观察凋亡相关蛋白细胞色素C （cyto C）、半胱氨酸天冬氨酸蛋白酶3（caspase3）和活化半胱氨酸天冬氨酸蛋白酶3（cleaved caspase3）蛋白表达水平，Muse^®凋亡试剂盒检测各组细胞凋亡率。结果：与对照组比较，CoCl₂组细胞存活率无明显变化（P>0.05），其余2组细胞存活率明显下降（P<0.05）；且联合组高于DDP组（P<0.05）。与对照组比较，CoCl₂组和联合组细胞中HIF-1α阳性表达强度明显增强（P<0.05）；DDP组和联合组细胞中iNOS阳性表达强度明显增强（P<0.05）。与对照组比较，DDP组和联合组细胞Ca²⁺水平升高（P<0.05）；且DDP组高于联合组（P<0.05）。与对照组比较，CoCl₂组细胞中cyto C、caspase3和cleaved caspase3蛋白表达水平无明显变化（P>0.05），DDP组细胞中cyto C、caspase3和cleaved caspase3蛋白表达水平明显升高（P<0.05）；且联合组低于DDP组（P<0.05）。与对照组比较，DDP组细胞凋亡率明显升高（P<0.05）；联合组细胞凋亡率较DDP组降低（P<0.05）。结论：CoCl₂可以通过抑制DDP诱导的人卵巢癌SKOV3细胞株iNOS表达增强来减少线粒体途径凋亡的发生，降低其顺铂敏感性。

Influence of CoCl_2 in cisplatin sensitivity of human ovarian cancer SKOV3 cells

Objective: To observe the effect of CoCl₂ on the cisplatin sensitivity of human ovarian cancer SKOV3 cells, and to clarify the possible mechanism.Methods: The SKOV3 cells were cultured in vitro and randomly divided into control group, CoCl₂ group, cisplatin (DDP) group and CoCl₂ combined with DDP(combination) group. The cells in CoCl₂ group were cultured in normal cell medium for 20 h after cultured in 200 μmol·L^-1 CoCl₂ for 4 h, the cells in DDP group were cultured in normal cell medium containing 10 mg·L^-1 DDP for 24 h, and the cells in combination group were cultured in 10 mg·L^-1 DDP for 20 h after cultured in 200 μmol·L^-1 CoCl₂ for 4 h. The survival rates of SKOV3 cells in various groups were detected by MTT method, and the positive expression intensities of hypoxia-inducible factor-1 α (HIF-1α) and inducible nitric oxide synthase (iNOS) in the cells in various groups were detected by immunofluorescence method. Rhod 2-AM fluorescence probe was used to observe the levels of Ca²⁺ in mitochondria in the cells in various groups. Western blotting method was used to observe the expression levels of cytochrome C (cyto C), cysteinyl aspartase 3 (caspase 3) and cleaved cysteinyl aspartase 3 (cleaved caspase 3). Muse^® apoptosis assay kit was used to detect the apoptotic rates of cells in various groups.Results: Compared with control group, the survival rate of the cells in CoCl₂ group had no significant change (P>0.05), and the survival rates of the cells in DDP and combination groups were decreased (P<0.05); the survival rate in combination group was higher than that in DDP group (P<0.05). Compared with control group, the positive expression intensities of HIF-1α in CoCl₂ and combination groups were increased (P<0.05). Compared with control group, the positive expressions of iNOS in DDP and combination groups were increased (P<0.05). The Ca²⁺ levels in the cells in DDP group and combination groups were higher than that in control group (P<0.05) and the Ca²⁺ level in DDP group was higher than that in combination group (P<0.05). Compared with control group, the expression levels of cyto C,caspase 3 and cleaved caspase 3 proteins in the SKOV3 cells in CoCl₂ group had no significant changes (P>0.05), and the expression levels of cyto C,caspase 3 and cleaved caspase 3 in DDP group were increased significantly(P<0.05); compared with DDP group, they were lower than those in combination group (P<0.05). Compared with control group, the apoptotic rate of SKOV3 cells in DDP group was increased significantly(P<0.05); the apoptotic rate of SKOV3 cells in combination group was lower than that in DDP group (P<0.05).Conclusion: CoCl₂ can redece the mitochondrial apoptosis of human ovarian cancer SKOV3 cells by inhibiting the DDP-induced enhancement of iNOS expression and decrease the sensitivity of SKOV3 cells to cisplatin.