前列腺凋亡反应基因4反义寡核苷酸抑制谷氨酸诱导的PC12细胞内钙离子浓度上调及其抗凋亡意义

Suppressive effects of par-4 antisense olig odeoxynucleotide on up-regulation of intracelluar calcium concentration in PC12 cell induced by glutamate and its anti-apoptosis effects

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 uppressive effects of par-4 antisense oligodeoxyn ucleotide on the up-regulation of intracellular calcium concentration in PC12 c ell induced by glutamate and its anti-apoptosis effects. Methods Cationic lipid-mediated par-4 antisense oligodeoxynucleotide (par-4-AS-ODN) was transfected into PC12 cells and followed by glutamate for treatment. Misma tch oligodeoxy-nucleotide (MS-ODN) was used as the control. Morphological ass essment and evaluation of the anti-apoptosis effects of par-4-AS-ODN on PC12 cells were performed by laser scanning confocal microscopy by double staining o f the cells with Hoechest 33258/propidium iodide (Hoe/PI) and flow cytometry res pectively. The mRNA and protein levels of calpain 10 and par-4 were measured b y RT-PCR and Western blot. Intracellular calcium concentration was determined by using laser scanning confocal microscope with Fura-2/AM as the fluorescent d ye. Results Par-4-AS-ODN repress the increase of par-4 protein in PC12 cell( 52.3±5.0 vs 90.0±3.2, P<0.01).Par-4-AS-ODN significantly inhibit ed the apoptosis of PC12 cells induced by glutamate(53% vs 31%, P<0.01). Par-4-AS-ODN significantly suppress the up-regulation of intracellular calc ium concentration in PC12 cells induced by glutamate(Rate of fluorescent densit y: 167.9±32.4 vs 228.8±36.8, P<0.01).Par-4-AS-ODN inhibited the increase of calpain 10 mRNA in PC12 cells induced by glutamate(46.3±3.7 vs 3 4.8±2.1, P<0.01). Conclusions par-4-AS-ODN enables to inhibit apoptosis of PC12 cells induced by glutamate. The mechanism of the inhibition may be closely related to suppressi on of the up-regulation of intracellular calcium concentration and calpain tran scription expression.