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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(7μmol·L-1尼莫地平或10μmol·L-1MK801)以及十六烷酸组(1、10、100μmol·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。
Objective To study the protective effect of palmitate on the apoptosis of PC12 cells induced by glutamate. Methods PC12 cells were divided into 7 groups: normal control group, dimethylsulfoxide solvent control group, model control group (25 mmol·L-1 glutamate), positive drug control group (7 μmol·L-1 nimodipine Or 10μmol·L-1MK801) and hexadecanoic acid group (1,10,100μmol·L-1). The protective effects of palmitate on PC12 cells injured by glutamate were examined by thiazolyl blue assay. The changes of membrane permeability of PC12 cells injured by glutamate were detected by lactate dehydrogenase colorimetric assay and Trypan blue staining Effects of AO / EB and Hoechst staining on PC12 cell apoptosis. FITC-Annexin V / PI fluorescent labeling flow cytometry apoptosis rate. Western blot was used to detect the expression of Bcl-2, Bax and GluR1. The results of thiazolyl blue, trypan blue staining, cell supernatant lactate dehydrogenase content showed: compared with the glutamate model group, hexadecanoic acid can significantly reduce glutamate damage PC12 cell viability and reduce cell membrane pass Permeability; AO / EB fluorescent staining showed glutamate damage, the model control group showed most of the cells stained AO stained nuclei chromatin showed concentrated staining, only a small number of palmitic acid groups were EB stained orange-red Cells; Hocehst staining showed that compared with the model group, palmitate group issued a weak blue fluorescence. Hexadecanoic acid can up-regulate the expression of Bcl-2 protein, inhibit the expression of Bax protein, and inhibit glutamate-induced glutamate receptor overexpression. Conclusion Hexadecanoic acid has a protective effect on the apoptosis of PC12 cells induced by glutamate, which is related to the inhibition of the increase of glutamate receptor protein NMDA, Bax protein expression and up-regulation of Bcl-2 protein expression.