摘要:
目的 初步探讨二甲基甲酰胺（dimethylformamide，DMF）是否通过线粒体通路致H9c2心肌细胞凋亡及可能机制。方法 H9c2心肌细胞体外培养，分组给予不同处理：正常对照组，N-乙酰半胱氨酸（N-acetyl-L-cysteine，NAC）（4 mmol/L）组，DMF（200 mmol/L）组，NAC（4 mmol/L）+DMF（200 mmol/L）组，24 h后使用免疫荧光细胞化学法（immune-fluorescence cytochemistry，IFC）检测细胞内细胞色素C（cytochrome C，Cyt-c）定位，蛋白免疫印迹法检测胞浆Cyt-c、Bcl-2、Cleaved caspse-3水平。结果 IFC观察发现，正常对照及NAC组Cyt-c呈点状或斑块状分布于胞浆，DMF作用24 h导致Cyt-c弥散分布于胞浆，而NAC可抑制DMF的这种作用；蛋白免疫印迹结果表明，Cyt-c蛋白水平组间差异具有统计学意义（F=26.14，P<0.001），与对照组相比，DMF组Cyt-c水平升高，NAC+DMF组较DMF组Cyt-c水平降低，差异均有统计学意义（均有P<0.001）；Bcl-2蛋白水平组间差异具有统计学意义（F=28.92，P<0.001），与对照组相比，DMF组Bcl-2蛋白水平降低，与DMF组相比，NAC+DMF组Bcl-2蛋白水平升高，差异均有统计学意义（均有P<0.001）；Cleaved caspase-3蛋白水平组间差异具有统计学意义（F=28.98，P<0.001）），与对照组相比，DMF组Cleaved caspase-3水平上升，NAC+DMF组较DMF组Cleaved caspase-3水平降低，差异均具有统计学意义（均有P<0.001）。结论 DMF可以通过线粒体通路诱导H9c2心肌细胞凋亡，氧化应激可能参与该通路的发生。
Abstract:
Objective To explore whether N, N-dimethylformamide (DMF) can induce apoptosis in H9c2 cardiomyocytes through mitochondrial pathway and its potential mechanism preliminarily. Methods H9c2 Cardiomyocytes were cultured in vitro and divided into groups with different treatments:control group, NAC group (4 mmol/L), DMF group (200 mmol/L), NAC (4 mmol/L)+DMF (200 mmol/L) group. After treating with 24 h, the location of Cyt-c was detected by immunofluorescence cytochemistry (IFC) and the levels of Cyt-c, Bcl-2, Cleaved caspase-3 were detected by western blotting. Results IFC showed that the control and NAC group of Cyt-c were distributed in spotted or patchy shape in the cytoplasm, and the DMF group of Cyt-c was distributed in the cytoplasm diffusely, while NAC could inhibit the effect induced by DMF. Western blotting showed that the levels of Cyt-c different groups were significantly different (F=26.14, P<0.001). Comparing with the control group, as for the level of Cyt-c, the DMF group was significantly increased and the NAC+DMF group was significantly decreased than DMF group (all P<0.001); as for the level of Bcl-2 among different groups were significantly different (F=28.92,P<0.001). Compare with control group, the DMF group was significantly decreased and the NAC+DMF group was significantly increased than DMF group (all P<0.001); as for the level of Cleaved caspase-3, there were significantly different (F=28.98,P<0.001) among different groups. Compare with control group, the DMF group was significantly increased and the NAC+DMF group was significantly decreased than DMF group (all P<0.001). Conclusions DMF can induce apoptosis in H9c2 Cardiomyocytes through mitochondrial pathway. Oxidative stress may be involved in the pathogenesis.
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