酒精对PC12细胞凋亡及神经鞘磷脂循环的影响
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摘要
目的本实验建立酒精诱导PC12细胞凋亡模型,应用此模型观察PC12细胞凋亡过程中神经鞘磷脂循环相关酶活性及mRNA表达量的改变,分析该循环在神经细胞凋亡中的作用,为进一步研究神经细胞的凋亡机制提供依据。
方法MTT法测定酒精对PC12细胞增殖的抑制作用；Hoechst33258染色荧光显微镜观察PC12细胞凋亡形态学变化；DNA琼脂糖凝胶电泳检测细胞凋亡梯状DNA条带；RT-PCR法检测酒精对PC12细胞SMS1、SMS2和N-SMase mRNA表达的影响；薄层层析方法测定SMS的活性；荧光分光光度法检测N-SMase的活性。
结果MTT法结果显示,去血清培养的PC12细胞24h,酒精浓度在100mmol/L、200 mmol／L、400 mmol/L和800 mmol/L时,细胞存活率分别是单纯去血清的87.54%、70.73%、57.89%和51.70%,表现出较强的细胞增殖抑制作用,与去血清对照组比较差异显著(P<0.05),呈浓度依赖性。含10%胎牛血清的细胞培养组,酒精浓度达到200mmol/L时对细胞的增殖抑制作用不明显,与正常培养组相比无显著性差异(P>0.05)。Hoechst 33258荧光染色观察细胞核形态学变化,结果显示,不同浓度的酒精作用PC12细胞24h,酒精处理组凋亡细胞增多,表现染色质凝集,细胞核变小、核碎裂成碎片等典型细胞凋亡特征性变化,凋亡率随着酒精浓度的增大而升高,去血清组的酒精浓度为100mmoL／L、200mmoL／L和300mmoL/L时,细胞凋亡率分别是19.21±3.2%(P<0.05)、28.39±5.11%(P<0.05)和38.68±4.28%(P<0.01),呈剂量依赖关系。琼脂糖凝胶电泳可见100-300mmoL／L浓度酒精处理组有不同程度的DNA断裂,显示凋亡细胞典型的梯状DNA。RT-PCR检测酒精对PC12细胞SMS和N-SMase基因在转录水平表达的影响,结果显示,不同浓度酒精作用于PC12细胞0.5h,SMS1表达量无显著变化,当作用时间达1h和2h, SMS1表达量显著增加,并呈剂量依赖性,而SMS2的mRNA表达则不受酒精作用的影响；不同浓度酒精作用PC12细胞0.5h和1h,N-SMase表达量无显著变化,作用2h时表达升高。以NBD-神经酰胺为底物,用薄层层析法检测细胞内总SMS活性,显示不同浓度酒精作用PC12细胞2h,SMS活性随酒精浓度增加而升高。荧光分光光度法检测N-SMase的活性,显示酒精作用PC12细胞0.5h时N-SMase活性变化不显著(P>0.05),当作用时间达2h时酶活性升高,与去血清组相比差异显著(P<0.05)。
结论：
1.酒精可导致PC12细胞凋亡,凋亡率与酒精浓度呈正相关。
2.酒精可致PC12细胞SMS1和N-SMase的mRNA表达量增高,酶活性增强。
Objective In this study, we established the ethanol-induced apoptotic model of pheochromocytoma (PC 12) cells. Using the model, we examined the key enzymes activity and mRNA expression changes of the sphingomyelin cycle pathway in PC 12 cells, and assessed the role of the sphingomyelin cycle pathway in apoptosis. Our data lend support to the further study of the neuron apoptosis mechanism.
Methods The inhibition effect of ethanol on PC 12 cells proliferation was examined by MTT assay. Cell apoptosis was determined by Hoechst 33258 fluorescence staining and DNA agarose gel electrophoresis mothod. The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the mRNA expression changes of SMS1, SMS2 and neutral sphingomyelin enzyme (N-SMase). The sphingomyelin synthase (SMS) activity was detected by the thin-layer chromatography, and N-SMase activity was detected by fluorescence spectrophotometry.
Results The ethanol with the concentration of 100 mmol/L,200 mmol/L,400mmol/L and 800 mmol/L decreased the number of PC12 cells to 87.54%、70.73%、57.89% and 51.70% of the control in serum-free media for 24 h. The result indicates the inhibition effect of ethanol on cell proliferation significantly in a dose-dependent manner (P<0.05). No change in viability was observed in cells exposed to 200 mmol/L ethanol in the presence of 10% serum (P>0.05). The nuclei morphological changes detected by using Hoechst 33258 staining and fluorescent microscopy. Addition of ethanol with different concentration to PC 12 cells, resulted in a number of morphological changes that are characteristic of apoptosis,these included cell shrinkage, chromatin compaction and nuclear fragmentation. After being treated with 100mmoL/L、200mmoL/L and 300mmoL/L ethanol in serum-free media for 24 h, the apoptosis rate was 19.21±3.2%(P<0.05),28.39±5.11% (P<0.05) and 38.68±4.28% (P<0.01). The data showed that the cell apoptosis rate was significantly increased by ethanol in does dependent manners. Agarose gel electrophoresis of the DNA isolated from ethanol(100-300mmoL/L)-treated cells maintained in the absence of serum yielded the classical DNA laddering pattern of 180 to 200 bp fragments suggestive of apoptosis. Treatment of PC 12 cells with different ethanol concerntration for 1 or 2 hours increased mRNA expression of SMS1 in does dependent manners,but did not increase mRNA expression of SMS2. After treated with different ethanol concerntration, total SMS activaty was increased in a dose-dependent manner. Although adding ethanol for 0.5-1 hours did not alter mRNA expression and activity of N-SMase, a significant increase in mRNA expression and activity of N-SMase was observed in PC 12 cells incubated for 2 hours in the presence of different concerntration ethanol.
Conclusions:
1. Ethanol can induce apoptosis of pheochromocytoma (PC 12) cells in a dose-dependent manner.
2. Treatment of PC 12 cells with different ethanol concerntration increased mRNA expression and activity of SMS1 and N-SMase in does dependent manners.
方法MTT法测定酒精对PC12细胞增殖的抑制作用；Hoechst33258染色荧光显微镜观察PC12细胞凋亡形态学变化；DNA琼脂糖凝胶电泳检测细胞凋亡梯状DNA条带；RT-PCR法检测酒精对PC12细胞SMS1、SMS2和N-SMase mRNA表达的影响；薄层层析方法测定SMS的活性；荧光分光光度法检测N-SMase的活性。
结果MTT法结果显示,去血清培养的PC12细胞24h,酒精浓度在100mmol/L、200 mmol／L、400 mmol/L和800 mmol/L时,细胞存活率分别是单纯去血清的87.54%、70.73%、57.89%和51.70%,表现出较强的细胞增殖抑制作用,与去血清对照组比较差异显著(P<0.05),呈浓度依赖性。含10%胎牛血清的细胞培养组,酒精浓度达到200mmol/L时对细胞的增殖抑制作用不明显,与正常培养组相比无显著性差异(P>0.05)。Hoechst 33258荧光染色观察细胞核形态学变化,结果显示,不同浓度的酒精作用PC12细胞24h,酒精处理组凋亡细胞增多,表现染色质凝集,细胞核变小、核碎裂成碎片等典型细胞凋亡特征性变化,凋亡率随着酒精浓度的增大而升高,去血清组的酒精浓度为100mmoL／L、200mmoL／L和300mmoL/L时,细胞凋亡率分别是19.21±3.2%(P<0.05)、28.39±5.11%(P<0.05)和38.68±4.28%(P<0.01),呈剂量依赖关系。琼脂糖凝胶电泳可见100-300mmoL／L浓度酒精处理组有不同程度的DNA断裂,显示凋亡细胞典型的梯状DNA。RT-PCR检测酒精对PC12细胞SMS和N-SMase基因在转录水平表达的影响,结果显示,不同浓度酒精作用于PC12细胞0.5h,SMS1表达量无显著变化,当作用时间达1h和2h, SMS1表达量显著增加,并呈剂量依赖性,而SMS2的mRNA表达则不受酒精作用的影响；不同浓度酒精作用PC12细胞0.5h和1h,N-SMase表达量无显著变化,作用2h时表达升高。以NBD-神经酰胺为底物,用薄层层析法检测细胞内总SMS活性,显示不同浓度酒精作用PC12细胞2h,SMS活性随酒精浓度增加而升高。荧光分光光度法检测N-SMase的活性,显示酒精作用PC12细胞0.5h时N-SMase活性变化不显著(P>0.05),当作用时间达2h时酶活性升高,与去血清组相比差异显著(P<0.05)。
结论：
1.酒精可导致PC12细胞凋亡,凋亡率与酒精浓度呈正相关。
2.酒精可致PC12细胞SMS1和N-SMase的mRNA表达量增高,酶活性增强。
Objective In this study, we established the ethanol-induced apoptotic model of pheochromocytoma (PC 12) cells. Using the model, we examined the key enzymes activity and mRNA expression changes of the sphingomyelin cycle pathway in PC 12 cells, and assessed the role of the sphingomyelin cycle pathway in apoptosis. Our data lend support to the further study of the neuron apoptosis mechanism.
Methods The inhibition effect of ethanol on PC 12 cells proliferation was examined by MTT assay. Cell apoptosis was determined by Hoechst 33258 fluorescence staining and DNA agarose gel electrophoresis mothod. The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the mRNA expression changes of SMS1, SMS2 and neutral sphingomyelin enzyme (N-SMase). The sphingomyelin synthase (SMS) activity was detected by the thin-layer chromatography, and N-SMase activity was detected by fluorescence spectrophotometry.
Results The ethanol with the concentration of 100 mmol/L,200 mmol/L,400mmol/L and 800 mmol/L decreased the number of PC12 cells to 87.54%、70.73%、57.89% and 51.70% of the control in serum-free media for 24 h. The result indicates the inhibition effect of ethanol on cell proliferation significantly in a dose-dependent manner (P<0.05). No change in viability was observed in cells exposed to 200 mmol/L ethanol in the presence of 10% serum (P>0.05). The nuclei morphological changes detected by using Hoechst 33258 staining and fluorescent microscopy. Addition of ethanol with different concentration to PC 12 cells, resulted in a number of morphological changes that are characteristic of apoptosis,these included cell shrinkage, chromatin compaction and nuclear fragmentation. After being treated with 100mmoL/L、200mmoL/L and 300mmoL/L ethanol in serum-free media for 24 h, the apoptosis rate was 19.21±3.2%(P<0.05),28.39±5.11% (P<0.05) and 38.68±4.28% (P<0.01). The data showed that the cell apoptosis rate was significantly increased by ethanol in does dependent manners. Agarose gel electrophoresis of the DNA isolated from ethanol(100-300mmoL/L)-treated cells maintained in the absence of serum yielded the classical DNA laddering pattern of 180 to 200 bp fragments suggestive of apoptosis. Treatment of PC 12 cells with different ethanol concerntration for 1 or 2 hours increased mRNA expression of SMS1 in does dependent manners,but did not increase mRNA expression of SMS2. After treated with different ethanol concerntration, total SMS activaty was increased in a dose-dependent manner. Although adding ethanol for 0.5-1 hours did not alter mRNA expression and activity of N-SMase, a significant increase in mRNA expression and activity of N-SMase was observed in PC 12 cells incubated for 2 hours in the presence of different concerntration ethanol.
Conclusions:
1. Ethanol can induce apoptosis of pheochromocytoma (PC 12) cells in a dose-dependent manner.
2. Treatment of PC 12 cells with different ethanol concerntration increased mRNA expression and activity of SMS1 and N-SMase in does dependent manners.
引文
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