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目的研究ATP敏感性钾通道(K)开放剂对缺血缺氧诱导PC12细胞凋亡及Akt蛋白和mRNA表达的影响,探讨KATP开放剂的保护作用机制。方法取传代后3dPc12细胞,分为对照组,缺血缺氧组,KATP通道开放剂组,KATP通道开放剂+阻断剂组。采用Annexin-vFITC/PI双染流式细胞分析仪检测凋亡率,应用免疫荧光染色和Western-blotting及RT-PCR方法检测p-Akt蛋白及mRNA表达水平。结果缺血缺氧组细胞凋亡率高于对照组,24h达高峰,KATP通道开放剂组低于缺血缺氧组,KATP通道开放剂+阻断剂组高于KATP通道开放剂组(P<0.01)。缺血缺氧后p-Akt蛋白及mRNA表达高于对照组,12h达高峰;KATP通道开放剂组高于缺血缺氧组,KATP通道开放剂+阻断剂组低于KATP通道开放剂组(P<0.01)。结论 KATP开放剂可能通过激活PI3K/Akt信号通路,对缺血后PC12细胞凋亡发挥保护作用。
Objective To investigate the effects of ATP-sensitive potassium channel openers on the apoptosis and the expression of Akt protein and mRNA in PC12 cells induced by hypoxia and hypoxia, and to explore the protective mechanism of KATP openers. Methods The passaged 3dPc12 cells were divided into control group, ischemia-hypoxia group, KATP channel opener group, KATP channel opener + blocker group. Annexin-vFITC / PI double staining flow cytometry was used to detect the apoptosis rate. The expression of p-Akt protein and mRNA was detected by immunofluorescence staining, Western-blotting and RT-PCR. Results The apoptotic rate of hypoxic-ischemic-hypoxic group was higher than that of the control group, reached the peak at 24 hours, the KATP channel opener group was lower than the hypoxic-ischemic group, the KATP channel opener + blocker group was higher than the KATP channel opener group (P <0.01). The expression of p-Akt protein and mRNA in hypoxic-ischemic and hypoxia groups were higher than those in control group and peaked at 12h. KATP channel opener group was higher than hypoxia-ischemia group, KATP channel opener + blocker group was lower than KATP channel opener group (P <0.01). Conclusion KATP opener may protect PC12 cells from apoptosis by activating PI3K / Akt signaling pathway.