酒精对PC12细胞凋亡及神经鞘磷脂循环的影响
发布时间：2018-08-20 07:40
【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(P0.05),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(P0.05)。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%(P0.05)、28.39±5.11%(P0.05)和38.68±4.28%(P0.01),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(P0.05),当作用时间达2h时酶活性升高,与去血清组相比差异显著(P0.05)。 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。
[Abstract]:Objective to establish PC12 cell apoptosis model induced by alcohol, observe the activity of neurilipid circulation-related enzymes and the expression of mRNA during apoptosis of PC12 cells, and analyze the role of this cycle in neuronal apoptosis. It provides evidence for further study of apoptosis mechanism of nerve cells. Methods the inhibitory effect of alcohol on the proliferation of PC12 cells was determined by MTT method. The morphological changes of apoptosis of PC12 cells were observed by fluorescence microscope with Hoechst33258 staining. DNA agarose gel electrophoresis was used to detect the ladder DNA bands of apoptosis. The effects of alcohol on the expression of SMS1, SMS2 and N-SMase mRNA in PC12 cells were detected by RT-PCR assay, the activity of SMS by TLC and the activity of N-SMase by fluorescence spectrophotometry. Results the results of MTT assay showed that the survival rate of PC12 cells cultured with serum free for 24 h and the concentration of alcohol at 100 mmol / L ~ 200 mmol / L for 400 mmol/L and 800 mmol/L were 87.54% and 57.89%, respectively, and 51.70%, respectively, showing a strong inhibitory effect on cell proliferation. Compared with the control group, the difference was significant (P0.05), in a concentration-dependent manner. In the culture group containing 10% fetal bovine serum, the inhibitory effect on cell proliferation was not obvious when alcohol concentration reached 200mmol/L, but there was no significant difference compared with the normal culture group (P0.05). Hoechst 33258 fluorescence staining was used to observe the morphological changes of the cell nucleus. The apoptosis rate of PC12 cells increased with the increase of alcohol concentration, chromatin agglutination, nuclear degeneration, nuclear fragmentation and fragmentation, when alcohol was treated with different concentration of alcohol for 24 h, and the apoptotic rate increased with the increase of alcohol concentration, and the apoptosis rate increased with the increase of alcohol concentration, and the apoptosis rate increased with the increase of alcohol concentration. In the serum free group, the apoptotic rate was 19.21 卤3.2% (P0.05) 28.39 卤5.11% (P0.05) and 38.68 卤4.28% (P0.01) in a dose-dependent manner. Agarose gel electrophoresis showed that there was different degree of DNA fragmentation in the alcohol treated group with 100-300mmoL/L concentration. The typical ladder DNA.RT-PCR of apoptotic cells was used to detect the effect of alcohol on the expression of SMS and N-SMase genes in PC12 cells at the transcriptional level. There was no significant change in the expression of SMS1 in PC12 cells treated with different concentrations of alcohol for 0.5 h. When the exposure time was 1 h and 2 h, the expression of SMS1 was significantly increased in a dose-dependent manner, while the mRNA expression of SMS2 was not affected by alcohol. The expression of N-SMase in PC12 cells did not change significantly at 0. 5 h and 1 h after exposure to different concentrations of alcohol, but increased at 2 h. The total SMS activity of PC12 cells was detected by thin layer chromatography with NBD- ceramide as substrate. The results showed that the activity of PC12 cells increased with the increase of alcohol concentration. The activity of N-SMase was detected by fluorescence spectrophotometry. The results showed that the N-SMase activity of PC12 cells did not change significantly at 0.5 h after alcohol treatment (P0.05), but the enzyme activity increased when the time reached 2 h, which was significantly different from that of serum removal group (P0.05). Conclusion: 1. Alcohol can induce apoptosis of PC12 cells, and the apoptotic rate is positively correlated with alcohol concentration. 2. 2. Alcohol could increase the mRNA expression of SMS1 and N-SMase and increase the activity of enzyme in PC12 cells.
【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363
本文编号：2192905
[Abstract]:Objective to establish PC12 cell apoptosis model induced by alcohol, observe the activity of neurilipid circulation-related enzymes and the expression of mRNA during apoptosis of PC12 cells, and analyze the role of this cycle in neuronal apoptosis. It provides evidence for further study of apoptosis mechanism of nerve cells. Methods the inhibitory effect of alcohol on the proliferation of PC12 cells was determined by MTT method. The morphological changes of apoptosis of PC12 cells were observed by fluorescence microscope with Hoechst33258 staining. DNA agarose gel electrophoresis was used to detect the ladder DNA bands of apoptosis. The effects of alcohol on the expression of SMS1, SMS2 and N-SMase mRNA in PC12 cells were detected by RT-PCR assay, the activity of SMS by TLC and the activity of N-SMase by fluorescence spectrophotometry. Results the results of MTT assay showed that the survival rate of PC12 cells cultured with serum free for 24 h and the concentration of alcohol at 100 mmol / L ~ 200 mmol / L for 400 mmol/L and 800 mmol/L were 87.54% and 57.89%, respectively, and 51.70%, respectively, showing a strong inhibitory effect on cell proliferation. Compared with the control group, the difference was significant (P0.05), in a concentration-dependent manner. In the culture group containing 10% fetal bovine serum, the inhibitory effect on cell proliferation was not obvious when alcohol concentration reached 200mmol/L, but there was no significant difference compared with the normal culture group (P0.05). Hoechst 33258 fluorescence staining was used to observe the morphological changes of the cell nucleus. The apoptosis rate of PC12 cells increased with the increase of alcohol concentration, chromatin agglutination, nuclear degeneration, nuclear fragmentation and fragmentation, when alcohol was treated with different concentration of alcohol for 24 h, and the apoptotic rate increased with the increase of alcohol concentration, and the apoptosis rate increased with the increase of alcohol concentration, and the apoptosis rate increased with the increase of alcohol concentration. In the serum free group, the apoptotic rate was 19.21 卤3.2% (P0.05) 28.39 卤5.11% (P0.05) and 38.68 卤4.28% (P0.01) in a dose-dependent manner. Agarose gel electrophoresis showed that there was different degree of DNA fragmentation in the alcohol treated group with 100-300mmoL/L concentration. The typical ladder DNA.RT-PCR of apoptotic cells was used to detect the effect of alcohol on the expression of SMS and N-SMase genes in PC12 cells at the transcriptional level. There was no significant change in the expression of SMS1 in PC12 cells treated with different concentrations of alcohol for 0.5 h. When the exposure time was 1 h and 2 h, the expression of SMS1 was significantly increased in a dose-dependent manner, while the mRNA expression of SMS2 was not affected by alcohol. The expression of N-SMase in PC12 cells did not change significantly at 0. 5 h and 1 h after exposure to different concentrations of alcohol, but increased at 2 h. The total SMS activity of PC12 cells was detected by thin layer chromatography with NBD- ceramide as substrate. The results showed that the activity of PC12 cells increased with the increase of alcohol concentration. The activity of N-SMase was detected by fluorescence spectrophotometry. The results showed that the N-SMase activity of PC12 cells did not change significantly at 0.5 h after alcohol treatment (P0.05), but the enzyme activity increased when the time reached 2 h, which was significantly different from that of serum removal group (P0.05). Conclusion: 1. Alcohol can induce apoptosis of PC12 cells, and the apoptotic rate is positively correlated with alcohol concentration. 2. 2. Alcohol could increase the mRNA expression of SMS1 and N-SMase and increase the activity of enzyme in PC12 cells.
【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363
【参考文献】
相关期刊论文 前1条
1 周涛,许百男,陈德蕙,阙海萍,林秋霞,吕双红,刘少君;PC12细胞分化的神经元与离体培养的大鼠原代皮质神经元之间功能性突触的形成[J];中华神经科杂志;2005年03期
，本文编号：2192905
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2192905.html
最近更新
教材专著
