地黄饮子对Aβ_（25-35）诱导PC12细胞损伤氧化应激及Ca~（2+）通道的影响
摘要
目的:本课题通过建立Aβ损伤的PC12细胞模型,运用生物化学和激光共聚焦技术,试图分析Aβ与AD发生、发展的密切关系及相关机制,探讨地黄饮子防治AD的作用机理。
方法:离体培养的PC12细胞分为6组:空白对照组、模型组、对照组、安理申组、地黄饮子低剂量组、地黄饮子中剂量组、地黄饮子高剂量组。待细胞进入对数生长期后吸去培养液,分别加入空白培养液10%、正常脑脊液10%、安理申脑脊液10%、中药脑脊液低剂量5%、中剂量10%、高剂量20%,加培养液补至等量, 37℃孵育2h。然后除空白组之外的各组加入4μL经老化处理的Aβ25-35 (终浓度为10μmol/L),建立AD细胞模型,空白组加入等量的培养液,继续孵育24h。然后收集培养液、离心细胞。对培养液进行MTT法和LDH的检测细胞生存活力;检测各组PC12细胞培养液中MDA、SOD、CAT、GSH-Px的含量;激光共聚焦法检测各组PC12细胞内钙离子与荧光探针结合后的荧光强度。
结果:一、地黄饮子能增强MTT代谢,减少LDH释放,改善Aβ25-35损伤后的PC12细胞生存状态,提高细胞生存活力;二、地黄饮子能降低Aβ25-35损伤PC12细胞培养液中的MDA含量,提高PC12细胞培养液中SOD的活性、提高CAT、GSH-Px的含量即抗氧化酶活性,提高自由基清除能力,减少自由基对PC12细胞的损伤;三、地黄饮子能抑制Aβ25-35损伤PC12细胞所致的钙超载现象,保护细胞膜结构;四、地黄饮子可能通过对细胞膜的保护作用,减少自由基对神经细胞的损害,进而提高细胞生存活力。
结论:地黄饮子可能通过抑制Aβ25-35损伤的PC12细胞所致的钙超载现象,减少LDH释放,改善细胞状态,进而提高抗氧化酶活性,增强自由基清除能力,减少自由基对神经细胞的损害,使细胞生存活力增强。
Objective: This study use chemical,biochemical and other advanced means and methods of detection to explore viability,the cell structure changes and the regulation of apoptosis of PC12 cells injured by Amyloidβ-protein and affected by the cerebrospinal fluid-Amyloid with DHYZ.Aβ25-35 PC12 cell injury model,the current international standards of AD model,was established and the neurotoxicity effect of apoptosis indeed by Aβ25-35 was confirmed.
Methods: The cultured cells of PC12 grown in the log phase were divided into 6 groups: normal control group,model control group,Aricept control group,low dose of DHYZ group,medium dose of DHYZ group and high dose of DHYZ group.The above 6 groups were respectively added 10% normal cultured fluid,10% normal cerebrospinal fluid,10% Aricept cerebrospinal fluid,5% DHYZ cerebrospinal fluid,10% and 20% dose DHYZ cerebrospinal fluid,in sequence which was then added cultured fluid to the equal standary.The abstained examples were hatched for 2 hours under 37℃.The same dose of cultured fluid were added to the normal group.The aged Aβ25-35 were added to the other five groups with the final concentration of 10 umol/L.All the whole six groups were hatched for 24 hours under 37℃.After centrifuging,the vitality of the cultured cells were measured with the methods of MTT and LDH. And detected the MDA, SOD, CAT,GSH-Px levels;also the intensity of calcium combined with fluorescence probe.
Result: The results showed: 1.DHYZ can enhance MTT metabolism rate, reduce the release of LDH.It can improve the cells survival activity after injured by Aβ25-35. 2.DHYZ could increase the SOD、CAT、GSH-Px levels and decrease the MDA levels in the cell culture fluid of PC12, increase the scavenging capacity of free radical.3.DHYZ can inhibit calcium overload, protect cell membrane structure.
Conclusion: DHYZ may inhibit calcium overload, reduce LDH release and enhance antioxidant enzyme activity of PC12 cells,which were injuried by aged Aβ25-35 ,DHYZ can improved cells state, increase free radical, clear the free radical to protect the cell vitality.
方法:离体培养的PC12细胞分为6组:空白对照组、模型组、对照组、安理申组、地黄饮子低剂量组、地黄饮子中剂量组、地黄饮子高剂量组。待细胞进入对数生长期后吸去培养液,分别加入空白培养液10%、正常脑脊液10%、安理申脑脊液10%、中药脑脊液低剂量5%、中剂量10%、高剂量20%,加培养液补至等量, 37℃孵育2h。然后除空白组之外的各组加入4μL经老化处理的Aβ25-35 (终浓度为10μmol/L),建立AD细胞模型,空白组加入等量的培养液,继续孵育24h。然后收集培养液、离心细胞。对培养液进行MTT法和LDH的检测细胞生存活力;检测各组PC12细胞培养液中MDA、SOD、CAT、GSH-Px的含量;激光共聚焦法检测各组PC12细胞内钙离子与荧光探针结合后的荧光强度。
结果:一、地黄饮子能增强MTT代谢,减少LDH释放,改善Aβ25-35损伤后的PC12细胞生存状态,提高细胞生存活力;二、地黄饮子能降低Aβ25-35损伤PC12细胞培养液中的MDA含量,提高PC12细胞培养液中SOD的活性、提高CAT、GSH-Px的含量即抗氧化酶活性,提高自由基清除能力,减少自由基对PC12细胞的损伤;三、地黄饮子能抑制Aβ25-35损伤PC12细胞所致的钙超载现象,保护细胞膜结构;四、地黄饮子可能通过对细胞膜的保护作用,减少自由基对神经细胞的损害,进而提高细胞生存活力。
结论:地黄饮子可能通过抑制Aβ25-35损伤的PC12细胞所致的钙超载现象,减少LDH释放,改善细胞状态,进而提高抗氧化酶活性,增强自由基清除能力,减少自由基对神经细胞的损害,使细胞生存活力增强。
Objective: This study use chemical,biochemical and other advanced means and methods of detection to explore viability,the cell structure changes and the regulation of apoptosis of PC12 cells injured by Amyloidβ-protein and affected by the cerebrospinal fluid-Amyloid with DHYZ.Aβ25-35 PC12 cell injury model,the current international standards of AD model,was established and the neurotoxicity effect of apoptosis indeed by Aβ25-35 was confirmed.
Methods: The cultured cells of PC12 grown in the log phase were divided into 6 groups: normal control group,model control group,Aricept control group,low dose of DHYZ group,medium dose of DHYZ group and high dose of DHYZ group.The above 6 groups were respectively added 10% normal cultured fluid,10% normal cerebrospinal fluid,10% Aricept cerebrospinal fluid,5% DHYZ cerebrospinal fluid,10% and 20% dose DHYZ cerebrospinal fluid,in sequence which was then added cultured fluid to the equal standary.The abstained examples were hatched for 2 hours under 37℃.The same dose of cultured fluid were added to the normal group.The aged Aβ25-35 were added to the other five groups with the final concentration of 10 umol/L.All the whole six groups were hatched for 24 hours under 37℃.After centrifuging,the vitality of the cultured cells were measured with the methods of MTT and LDH. And detected the MDA, SOD, CAT,GSH-Px levels;also the intensity of calcium combined with fluorescence probe.
Result: The results showed: 1.DHYZ can enhance MTT metabolism rate, reduce the release of LDH.It can improve the cells survival activity after injured by Aβ25-35. 2.DHYZ could increase the SOD、CAT、GSH-Px levels and decrease the MDA levels in the cell culture fluid of PC12, increase the scavenging capacity of free radical.3.DHYZ can inhibit calcium overload, protect cell membrane structure.
Conclusion: DHYZ may inhibit calcium overload, reduce LDH release and enhance antioxidant enzyme activity of PC12 cells,which were injuried by aged Aβ25-35 ,DHYZ can improved cells state, increase free radical, clear the free radical to protect the cell vitality.
引文
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