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目的 研究前列腺凋亡反应基因4(par -4)反义寡核苷酸拮抗谷氨酸对PC12细胞内游离钙离子浓度的上调作用及其抗凋亡意义。方法 脂质体介导转染par -4反义寡核苷酸。谷氨酸诱导PC12细胞凋亡。Hoechest33258 /碘化丙啶荧光染色观察细胞凋亡形态,流式细胞术分析凋亡百分率。Fura 2 /AM荧光染色结合激光共聚焦显微镜测定细胞内游离钙离子浓度。逆转录聚合酶链反应(RT- PCR)测定钙依赖性蛋白酶Calpain10的mRNA水平。Western印迹测定par -4蛋白表达量。结果与正常对照组(25 .6±4.1 )相比,谷氨酸诱导PC12细胞中par- 4蛋白表达上调( 90. 0±3 2,P<0. 01),par- 4反义寡核苷酸拮抗其上调( 52 .3±5 .0,P<0 .01 );谷氨酸诱导凋亡组凋亡百分率为53%, par -4反义寡核苷酸拮抗谷氨酸诱导的PC12细胞凋亡(31%,P<0. 01);谷氨酸诱导PC12细胞内游离钙离子浓度上调,par- 4反义寡核苷酸拮抗其上调(荧光强度比值分别为167 .9±32 .4、228. 8±36. 8,P<0 .01);谷氨酸诱导PC12细胞内钙依赖性蛋白酶Calpain10mRNA水平上调( 46 .3±3 .7 ),par- 4反义寡核苷酸抑制其上调(34 8±2 1,P<0 01 )。结论 par -4反义寡核苷酸拮抗谷氨酸诱导的PC12细胞凋亡,其机制可能与抑制细胞内游离钙离子浓度上调和抑制Calpain10基因转录有关。
Objective To investigate the effect of anti-apoptotic gene 4 (par-4) antisense oligonucleotide on the upregulation of intracellular free calcium concentration in PC12 cells and its anti-apoptotic effect. Methods Liposome-mediated transfection of par-4 antisense oligonucleotide. Glutamate Induces PC12 Cell Apoptosis. Hoechest 33258 / propidium iodide fluorescence staining observed apoptotic morphology, flow cytometry analysis of apoptosis percentage. Fura 2 / AM fluorescence staining combined with confocal laser scanning microscopy intracellular free calcium concentration. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine the mRNA level of Calpain10, a calcium-dependent protease. Western blot was used to measure the expression of par-4 protein. Results Compared with the normal control group (25.6 ± 4.1), glutamate induced up-regulation of par-4 protein expression in PC12 cells (90.0 ± 3.2, P <0.01) (52.3 ± 5.0, P <0.01). The percentage of apoptotic cells in glutamate-induced apoptosis group was 53%, while that of par-4 antisense oligonucleotide antagonized glutamate-induced PC12 cell apoptosis (31%, P <0.01); glutamate-induced up-regulation of intracellular free calcium concentration in PC12 cells and up-regulation of par-4 antisense oligonucleotides (fluorescence intensity ratio of 167.9 ± 32.4,228.8 ± 36.8, P <0.01). Glutamate induced up-regulation of calpain10mRNA in PC12 cells (46.3 ± 3.77) Glypican inhibited its up-regulation (34 8 ± 21, P <0.01). Conclusion Par-4 antisense oligonucleotide antagonizes glutamate-induced apoptosis in PC12 cells, which may be related to the inhibition of intracellular free calcium concentration and the inhibition of Calpain10 gene transcription.