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目的研究par-4反义寡核苷酸拮抗谷氨酸对PC12细胞中ERK1/2活性的下调作用及其抗凋亡作用。方法利用脂质体将par4反义寡核苷酸转染入PC12细胞。谷氨酸诱导PC12细胞凋亡。利用吖啶橙/溴化乙锭荧光染色观察PC12细胞形态,流式细胞分析评价凋亡百分率。Western印迹测定par4的蛋白质表达量和磷酸化的ERK1/2的蛋白表达量。结果(1)谷氨酸诱导PC12细胞中par-4蛋白表达上调,Par4反义寡核苷酸呈剂量依赖性地拮抗其上调(组间比较,P<0.01)。(2)谷氨酸诱导PC12细胞中磷酸化(Thr202/Tyr204)的ERK1/2蛋白水平下调,par-4反义寡核苷酸拮抗其上调(组间比较,P<0.01)。(3)Par-4反义寡核苷酸拮抗谷氨酸诱导的PC12细胞凋亡,如予ERK1/2阻断PD98059预处理,则其拮抗作用被下调(组间比较,P<0.05)。结论Par-4反义寡核苷酸拮抗谷氨酸诱导的PC12细胞凋亡,其机制可能与ERK的活化有关。
Objective To investigate the down-regulation of ERK1 / 2 activity by PC-4 antisense oligonucleotide and its anti-apoptotic effects in human PC12 cells. Methods The antisense oligonucleotides of par4 were transfected into PC12 cells by liposome. Glutamate Induces PC12 Cell Apoptosis. The morphology of PC12 cells was observed by acridine orange / ethidium bromide staining, and the percentage of apoptosis was evaluated by flow cytometry. Western blot was used to determine the protein expression of par4 and the protein expression of phosphorylated ERK1 / 2. Results (1) Glutamate up-regulated the expression of par-4 protein in PC12 cells. Par4 antisense oligonucleotide antagonized its up-regulation in a dose-dependent manner (comparison between groups, P <0.01). (2) Glutamate induced downregulation of ERK1 / 2 phosphorylation (Thr202 / Tyr204) in PC12 cells and upregulation of par-4 antisense oligonucleotides (comparison between groups, P <0.01). (3) Par-4 antisense oligonucleotide antagonized glutamate-induced apoptosis in PC12 cells. When ERK1 / 2 was blocked by PD98059 pretreatment, its antagonism was down-regulated (compared between groups, P <0.05). Conclusion Par-4 antisense oligonucleotide antagonizes glutamate-induced apoptosis of PC12 cells, which may be related to the activation of ERK.