H_2O_2对PC12细胞Caspase-3和NF-κB P65基因表达的影响

Effects of H_2O_2 on the Expression of Gene Caspase-3 and NF-ΚB P65 in the PC12 cell

目的 :探讨H2 O2 对PC12细胞半胱天冬酶 3(caspase 3)和核转录因子 kappaBP6 5 (uclearfactor kappaB ,NF κBP6 5 )基因表达的影响。方法 :不同剂量H2 O2 (0～ 4 0 0nmol·L-1)处理PC12细胞 8h ,采用RT PCR检测Caspase 3、NF κBP6 5mRNA表达变化 ,Westernblot检测Caspase 3P 2 0活性片段和NF κBP6 5蛋白表达的变化 ,用比色法检测Caspase 3相对活性。结果 :随H2 O2 浓度从 10 0～ 4 0 0nmol·L-1增加 ,Caspase 3、NF κBP6 5mRNA表达和Caspase 3P2 0活性片段及NF κBP6 5蛋白表达水平逐渐增加 ;随H2 O2 浓度从 5 0～ 4 0 0nmol·L-1增加 ,Cas pase 3相对活性增加 (P <0 .0 5 ) ,在 4 0 0nmol·L-1H2 O2 时Caspase 3相对活性达最大值。 结论 :H2 O2 可剂量依赖性的增加Caspase 3mRNA表达和Caspase 3P 2 0活性片段 ,增加Caspase 3活性 ;H2 O2 可剂量依赖性的增加NF κBP6 5mRNA表达和NF κBP6 5蛋白表达。

Objective: To determine the effects of H 2 O 2 on the expression of gene Caspase-3 and NF-κB P65 in PC12 cells. Methods: After the PC12 cells were treated with H 2 O 2 (0～400 nmol·L -1 ) for 8 h, mRNA expression of caspase-3 and NF-κB P65 were detected by RT-PCR, protein expression of caspase-3P 20 and NF-κB P65 were detected by Western blot, and caspase-3 relative activities(A 405 ) was determined by colormetric assay. Results: The mRNA and protein expression of Caspase-3 and NF-κB P65 gene were upregulated with 100～400 nmol·L -1 H 2 O 2 . The activity of Caspase-3 was increased with 50～400 nmol·L -1 H 2 O 2 (P<0.05), and it reached the peak in 400 nmol·L -1 H 2 O 2 group. Conclusion: H 2 O 2 increased the mRNA and protein expression of caspase-3 gene in a dose-dependent manner. The mRNA and protein expression of NF-κB P65 gene were upregulated by H 2 O 2 in a dose-dependent manner, but not in a time-dependent manner. It also increased the caspase-3 relative activities in a dose-dependent manner.