Par-4基因在PC12细胞缺氧后的表达及其反义寡核苷酸的抗凋亡作用

The expression of Par-4 gene in PC12 cells after hypoxia injury and anti-apoptosis effect of Par-4 antisense oligodeoxynucleotide

目的研究前列腺凋亡反应基因4(Par4)在PC12细胞缺氧后的表达及其反义寡核苷酸(ASODN)的抗凋亡作用。方法在正常或缺氧条件下培养PC12细胞,分组将不同浓度Par4反义寡核苷酸转染PC12细胞,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。Westernblot测定Par4蛋白表达量,比色法检测Caspase3的酶活性。结果缺氧24h,PC12细胞中Par4蛋白表达量上调157.42±7.53,明显高于正常对照组10.51±3.36(P<0.01)。10μmol/L的Par4,ASODN使Par4蛋白降为31.79±3.09,明显低于缺氧组157.42±7.53(P<0.01)。Par4的ASODN抑制缺氧诱导的PC12细胞凋亡,10μmol/LASODN使细胞凋亡百分数降为(34.5±3.5)%,明显低于缺氧组的(69.3±5.7)%(P<0.05)。Par4的ASODN抑制缺氧诱导的PC12细胞中Caspase3的酶活性上调,10μmol/LASODN使其活性降为0.549±0.078,与缺氧组0.917±0.051相比,差别有显著性意义(P<0.05)。结论Par4基因可能参与了缺氧诱导的PC12细胞损害。Par4ASODN可拮抗缺氧诱导的PC12细胞凋亡,其机制可能与Caspase3酶活性下调有关。

Objective To investigate the effects of hypoxia on the expression of prostate apoptosis response-4(Par-4) gene in PC12 cells,and anti-apoptosis effect of Par-4 antisense oligodeoxynucleotide(AS-ODN).Methods PC12 cells were cultured in normal or hypoxic condition.Cationic lipid-mediated AS-ODN was transfected into PC12 cells before hypoxia.And mismatch ODN (MS-ODN) was also transfected into the cells as control.Morphological observation and the detection of anti-apoptosis effects of Par-4 AS-ODN on PC12 cells were done with laser scanning confocal microscope by double staining the cells with acridine orange/ethidium bromide(AO/EB). Percentage of apoptosis was evaluated with flow cytometry.The protein eexpression of Par-4 was determined by Western blot.Caspase-3 relative activity was detected by colormetric assay.Results Compared with that of normal control group,the protein level of Par-4 in PC12 cells was significantly increased after hypoxia for 12 hours(157.42±7.53 vs 10.51±3.36,P<0.01).Par-4 AS-ODN inhibited the protein expression of Par-4 in PC12 cells after hypoxia 24 hours in a dose-dependent manner.The Par-4 protein in hypoxia group reached 157.42±7.53,and 10μmol/L AS-ODN can significantly down-regulat the Par-4 protein expression(31.79±3.09, P<0.01).Par-4 AS-ODN could inhibit the apoptosis of PC12 cells after hypoxia for 24 hours.Transfection of 10μmol/L AS-ODN made apoptotic rate decreased to (34.5±3.5)% from (69.3±5.7)%(P<0.05).Par-4 AS-ODN inhibited the increase of Caspase-3 relative activity in PC12 cells exposed to hypoxia for 24 hours(0.549±0.078 vs 0.917±0.051,P<0.05).Conclusion Par-4 gene might be involved in the damage of PC12 cells after hypoxia.Par-4 AS-ODN could inhibit the apoptosis of PC12 cells exposed to hypoxia.Its mechanism may be related to the inhibition of activation of Caspase-3.