【摘要】 目的:观察胞外高浓度ATP损伤人神经母细胞瘤SH-SY5Y细胞的作用,探讨损伤中自噬和凋亡发生的规律和特点。方法:培养的SH-SY5Y细胞按照加入ATP的浓度和作用时间进行分组。CCK-8法检测细胞生存率,单丹磺酰戊二胺染色检测自噬空泡的变化,Hoechst 33258染色检测细胞凋亡,流式细胞术检测细胞凋亡率变化,蛋白质印迹法检测半胱氨酸天冬氨酸蛋白酶3(caspase-3)及微管相关蛋白1轻链3-Ⅱ(microtubule-associated protein 1 light chain 3-Ⅱ,LC3-Ⅱ)的表达。结果:(1)与对照组相比,不同浓度(3、6、9、12、15 mmol/L)的ATP作用3 h均使SH-SY5Y细胞存活率明显降低,且呈剂量依赖性;不同作用时间(1、2、3、6 h)ATP(6 mmol/L)也可使SHSY5Y细胞存活率明显降低,3 h达高峰,呈时间依赖性。(2)ATP作用1 h时SH-SY5Y细胞自噬空泡显著增多(P<0.05),随时间延长,6 h时降到对照水平;ATP作用1 h时,LC3-Ⅱ表达显著增强(P<0.05),形成高峰,与自噬空泡增多的时点重合,2 h、3 h时LC3-Ⅱ表达水平逐渐减弱,6 h时降到对照水平。(3)与对照组相比,ATP作用3 h后细胞凋亡率达高峰(P<0.05),6 h时仍然保持在3 h的水平;cleaved caspase-3表达量同步增强(P<0.05),6 h时达高峰。结论:胞外高浓度ATP能够诱导SH-SY5Y细胞自噬和凋亡;自噬增强在前,凋亡高潮在后;随着ATP作用时间的延长,凋亡占主导地位。更多还原

【Abstract】 AIM: To examine the effects of high concentration of extracellular ATP on human neuroblastoma SH-SY5 Y cell injury. METHODS: Cultured SH-SY5 Y cells were grouped according to the concentrations of ATP and treatment time. The cell viability was detected by CCK-8 assay. The variation of autophagic vacuoles was observed with monodansylcadaverine staining. The cell apoptosis was analyzed by Hoechst 33258 staining. Meanwhile,apoptotic rate was detected by flow cytometry. The levels of caspase-3 and microtubule-associated protein 1 light chain 3-Ⅱ( LC3-Ⅱ) were determined by Western blotting. RESULTS: Compared with control group,the survival rate of SH-SY5 Y cells was significantly reduced by ATP at different concentrations( 3,6,9,12 and 15 mmol /L for 3 h) and different treatment time( 1,2,3 and 6 h with 6 mmol /L ATP,peaking at 3 h). The autophagic vacuoles of SH-SY5 Y cells were significantly increased at 1 h with ATP treatment,trended to decrease over time and returned to control level at 6 h. The protein expression of LC3-Ⅱ was significantly increased at 1 h with ATP treatment,which was consistent with the time points of increasing autophagic vacuoles. LC3-Ⅱ expression level gradually decreased at 2 ~ 3 h with ATP treatment,and returned to control level at 6 h. Compared with control group,the apoptotic rate and the expression level of caspase-3 were enhanced synchronously.The peak of apoptotic rate occurred at 3 h,and kept until 6 h. The level of cleaved caspase-3 expression peaked at 6 h.CONCLUSION: High concentration of extracellular ATP induces the autophagy and apoptosis of SH-SY5 Y cells. The increased autophagy shows up,followed by the climax of apoptosis until 6 h. With the prolonged duration of ATP,apoptosis is the main process in the cells.更多还原