UVB诱导A375细胞自噬与凋亡调控机制探讨

Regulatory mechanism of UVB-induced autophagy and apoptosis in A375 cells

目的探究中波紫外线(UVB)对人表皮黑色素瘤细胞株A375细胞自噬与凋亡的诱导效应。方法 (1)取对数生长期A375细胞,分别予剂量为10.0、15.0 m J/cm^2的UVB照射,于照射后3、6、9、12 h收集细胞,用单丹磺酰戊二胺染色法观察细胞自噬,用吖啶橙/溴乙锭染色法观察细胞凋亡。(2)予相应剂量的UVB照射10.0、15.0 m J/cm^2组A375细胞,于照射后18、24、36、48 h收集细胞,以CCK-8法检测细胞存活率。(3)予剂量为10.0、15.0 m J/cm^2的UVB照射A375细胞,于照射后3、6、9、12 h收集细胞;予剂量为2.5、5.0、7.5、10.0、15.0 m J/cm^2的UVB照射A375细胞,于照射后9 h收集细胞;用免疫印迹法检测B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、Bcl-2相互作用蛋白(Beclin-1)、微管相关蛋白1轻链3(LC3)Ⅱ的表达。上述3个实验均另设不予UVB照射(予假照射)的对照组。结果 (1)剂量为10.0、15.0 m J/cm^2的UVB照射诱导的A375细胞自噬和细胞凋亡均表现为随着照射后时间的延长而增强,但在15.0 m J/cm^2UVB照射后12 h,细胞自噬已观察到降低。(2)10.0、15.0 m J/cm^2组细胞活力均随照射后时间的推移而增加(P<0.05);在照射后18~48 h,10.0和15.0 m J/cm^2组细胞活力均低于对照组(P<0.05);在照射后24~48 h,15.0 m J/cm^2组细胞活力均低于10.0 m J/cm^2组(P<0.05)。(3)10.0 m J/cm^2组细胞Beclin-1和LC3Ⅱ蛋白相对表达水平在照射后0~12 h内均随照射时间的增加而增加(P<0.05),而15.0 m J/cm^2组细胞仅在照射后0~9 h内观察到上述改变,在12 h时间点上述2个自噬相关蛋白明显下降(P<0.05);10.0和15.0 m J/cm^2组细胞Bcl-2蛋白相对表达水平在照射后3~12 h内均随照射时间的增加而下降(P<0.05),Bax蛋白相对表达水平均在照射后0~12 h内随照射时间的增加而增加(P<0.05)。0.0~10.0 m J/cm^2组细胞Beclin-1和LC3Ⅱ蛋白相对表达水平,0.0~15.0 m J/cm^2组细胞Bax蛋白相对表达水平,均随照射剂量的增加而增加(P<0.05);5.0~15.0 m J/cm^2组Bcl-2蛋白相对表达水平随照射剂量的增加而下降(P<0.05)。结论剂量为10.0和15.0 m J/cm^2UVB照射均可诱导A375细胞发生自噬和凋亡;10.0 m J/cm^2UVB照射诱导的细胞自噬可部分抵抗UVB对凋亡的诱导而使细胞活力增强,15.0 m J/cm^2UVB诱导的自噬不足以发挥上述作用,且以诱导凋亡为主导效应。

Objective To investigate the effect of ultraviolet B（ UVB） on autophagy and apoptosis in human epidermal melanoma A375 cells. Methods i） A375 cells at logarithmic growth phase were exposed to UVB at doses of 10. 0 and15. 0 m J/cm2. Then cells were collected at time point of 3,6,9 and 12 hours after irradiation. The effect of UVB on cell autophagy was observed by monodansylcadaverine staining and the effect of UVB on cell apoptosis was observed by acridine orange/ethidium bromide staining. ii） A375 cells of 10. 0 m J/cm2 group and 15. 0 m J/cm2 group were exposed to corresponding dose of UVB irradiation. Then cells were collected at time point of 18,24,36 and 48 hours after irradiation,and cell survival rate was examined using CCK-8 assay. iii） A375 cells were irradiated with UVB at doses of 10. 0 and15. 0 m J/cm2 and then cells were collected at time point of 3,6,9 and 12 hours after irradiation. After that,A375 cells were irradiated at doses of 2. 5,5. 0,7. 5,10. 0 and 15. 0 m J/cm2 of UVB,then cells were collected at time point of 9 hours after irradiation. The expressions of B-lymphoblastoma-2（ Bcl-2）,Bcl-2 related X protein（ Bax）,Bcl-2 interacting protein（ Beclin-1） and microtubule-associated protein 1 light chain 3（ LC3） Ⅱ were detected by Western blotting. A375 cells with no UVB irradiation were set as the control（ pseudo-irradiation） in each experiment. Results i） Both autophagy and apoptosis of A375 cells induced by UVB irradiation at doses of 10. 0 and 15. 0 m J/cm2 increased with time after irradiation. The effect on autophagy decreased at 12 hours time point with 15. 0 m J/cm2 UVB irradiation. ii） The cell viability increased with time after irradiation in the 10. 0 and 15. 0 m J/cm2 groups（ P〈0. 05）. From 18-48 hours after irradiation,the cell viability of the 10. 0 and 15. 0 m J/cm2 groups was lower than that of the control group（ P〈0. 05）.From 24-48 hours after irradiation,the cell viability of the 15. 0 m J/cm2 group was lower than that of the 10. 0 m J/cm2 group（ P〈0. 05）. iii） The relative expression of Beclin-1 and LC3 Ⅱ protein at the 10. 0 m J/cm2 group increased with time after 0-12 hours irradiation（ P〈0. 05）. The above changes of the 15. 0 m J/cm2 group were observed within 0 to 9 hours,and the above two autophagy-related proteins were significantly decreased at the 12 hours time point（ P〈0. 05）.The relative expression of Bcl-2 protein at the 10. 0 and 15. 0 m J/cm2 groups decreased with increasing time from 3 to 12 hours after irradiation（ P〈0. 05）. The relative expression of Bax protein increased with time from 0 to 12 hours after irradiation（ P〈0. 05）. The relative expression of Beclin-1 and LC3 protein in cells at 0. 0-10. 0 m J/cm2,and the relative expression of Bax protein in cells at 0. 0-15. 0 m J/cm2 increased with increase of irradiation dose（ P〈0. 05）. The relative expression of Bcl-2 protein decreased with increase of irradiation dose at 5. 0-15. 0 m J/cm2（ P〈0. 05）. Conclusion Autophagy and apoptosis of A375 cells can be induced by UVB irradiation at doses of 10. 0 and 15. 0 m J/cm2. Autophagy induced by UVB irradiation at 10. 0 m J/cm2 partially resisted the induction of apoptosis by UVB and enhanced cell viability. 15. 0 m J/cm2 UVB-induced autophagy was insufficient to exert the above-mentioned effects,and the induction of apoptosis was the dominant effect.