目的 研究十六烷酸对谷氨酸损伤PC12细胞凋亡的保护作用。方法 将PC12细胞分为7组:正常对照组,二甲基亚砜溶剂对照组,模型对照组(25 mmol·L-1谷氨酸),阳性药物对照组(7 μmol·L-1尼莫地平或10 μmol·L-1MK801)以及十六烷酸组(1、10、100 μmol·L-1)。噻唑蓝法验证十六烷酸对谷氨酸损伤PC12细胞保护作用;乳酸脱氢酶比色法、台盼蓝染色法检测不同浓度十六烷酸对谷氨酸损伤PC12细胞膜通透性变化的影响;AO/EB、Hoechst荧光染色法观察PC12细胞凋亡的形态。FITC-Annexin V/PI荧光标记流式细胞仪检测细胞凋亡率。Western blot法检测细胞内Bcl-2、Bax、GluR1蛋白表达。结果 噻唑蓝、台盼蓝染色计数、细胞上清液中乳酸脱氢酶含量显示:与谷氨酸模型组相比,十六烷酸能明显改善谷氨酸损伤PC12细胞的活力,减少细胞膜通透性;AO/EB荧光染色显示谷氨酸损伤后,模型对照组可见大部分细胞经AO染色后核染色质部分呈现浓染,十六烷酸各组仅出现少数被EB染成橘红色的细胞;Hocehst荧光染色显示,与模型组相比十六烷酸组细胞核发出蓝色荧光较弱。十六烷酸能上调Bcl-2蛋白表达,抑制Bax蛋白表达;并能抑制谷氨酸导致的谷氨酸受体的过度表达。结论 十六烷酸对谷氨酸损伤PC12细胞凋亡具有保护作用,该保护与抑制谷氨酸受体蛋白NMDA、Bax蛋白的表达的增加,上调Bcl-2蛋白表达等有关。
Abstract
OBJECTIVE To explore the protective effect of palmitic acid against glutamate-induced apoptosis in PC12 cells. METHODS PC12 cells were randomly divided into six groups: normal group, solvent group(DMSO 0.1%), model group (Glu 25 mmol·L-1), nimodipine(NMP,7 μmol·L-1)-treated group or MK801(10 μmol·L-1) -treated group set as positive control group, palmitic acid groups of 100, 10, 1 μmol·L-1 dose. Cell viability and membrane permeability were examined by MTT assay and LDH kit. Morphologic change of apoptosis were detected by AO/EB and Hoechst staining with fluorescence microscope. The apoptosis rates were measured by flow cytometry using fluorescein labeling FITC-Annexin V/PI. The expression of Bcl-2, Bax and GluR1 protein was assayed by Western blot methods. RESULTS Compared with the model group, the results showed that palmitic acid could significantly improve the viability of PC12 cells damaged by glutamate, and reduce the permeability of cell membranes. After AO/EB staining, nuclear chromatin of the cells in model group were stained strongly into orange by EB. In palmitic acid –treated group, only few cells were stained into orange. Compared with model group, after the treatment of palmitic acid , slight blue fluroscence stained by Hoechst in nucleus had been emitted. Palmitic acid could increase the expression of Bcl-2 and inhibit the level of the expression of Bax protein and GluR1 protein. CONCLUSION In summary, palmitic acid had protective effect on glutamate-induced apoptosis in PC12 cells through increasing the expression of Bcl-2 and inhibiting the expression of Bax and GluR1.
关键词
十六烷酸 /
PC12细胞 /
谷氨酸损伤 /
凋亡 /
保护作用
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Key words
:palmitic acid /
PC12 cells /
glutamic acid /
apoptosis /
protection
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中图分类号：
R965
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参考文献
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脚注
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基金
国家自然科学基金资助项目(81374005);十二五科技支撑计划资助项目(2012BAI26B03);安徽省第五批优秀青年科技基金资助项目(10040606Y17);2013年度安徽省学术及技术带头人及后备人选科研活动经费资助项目
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