【作者】 曹忠平；

【导师】 高钰琪；

【作者基本信息】 第三军医大学 ， 病理生理学， 2009， 博士

【摘要】 前言活性氧包括超氧阴离子(O2.-)、过氧化氢(H2O2)、羟自由基(.OH)和其它氧化物,过去认为是有细胞毒性的,能够损害脂质、蛋白和DNA。除外这些有害作用外,相当多的证据表明许多刺激因素能够刺激产生小量活性氧,该活性氧可作为对这些刺激反应的第二信使。以前认为缺血预处理(IPC)能够保护组织免受损伤。缺血预处理已经作为生物界的一个普遍的细胞保护机制。现在预处理的概念已经扩展到非缺血性应急反应,如活性氧预处理。许多刺激可导致活性氧的产生,从而上调细胞外信号调节激酶(ERK)的活性。而且,ERK可以通过激活一系列转录因子而促进B细胞淋巴瘤白血病2(Bcl-2)基因的表达。Bcl-2蛋白家族在调节程序性死亡过程中发挥重要的作用。本文旨在研究35%氧气预处理3小时是否能够保护1%氧气诱导的PC12毒性,35%氧气预处理诱导的细胞保护作用是否和活性氧产生,细胞外信号激酶途径及Bcl-2过表达有关。方法PC12细胞培养液为DMEM液。PC12细胞先用35%O2预处理180min,然后恢复12小时,最后1%O2低氧暴露72小时。在35%O2预处理前培养液换为无血清培养液。相应药物加入时间为35%O2预处理前60min。细胞活力用MTT法测定。细胞内ROS检测采用对过氧化物敏感的荧光探针DCFH-DA和HEt。PC12细胞用脂质体2000和SiRNA按说明书操作转染。ERKmRNA分析采用RT-PCR法。ERK、Bcl-2表达采用Western Blot。结果1. 35%O2预处理对低氧诱导的PC12细胞的影响1%氧气能导致相当数量的PC12死亡。但该细胞毒性作用能被35%O2预处理3小时恢复12小时逆转(P<0.01),而随后的0、24小时恢复无此作用。2. ROS产生PC12在35%O2和pyrogallol组相比21%O2组能够产生更多O2.?,而tempol组产生的O2.?明显少于35%O2组。35%O2预处理产生的H2O2相比21%O2预处理组无明显增多。细胞活力在pyrogallol和35%O2组明显增强,而在tempol组明显减弱。3. ERK表达35%O2预处理可以明显增强细胞活力和磷酸化的ERK1/2表达,这些效应可被ERK信号通路阻止剂PD98059而非PKA/PKC和PI3k/Akt通路阻止剂H7和wortmannin明显减弱。35%O2诱导的磷酸化ERK1/2的过表达可被4-羟-tempol明显减弱。用21%O2和20μMpyrogallol预处理同样能够诱导磷酸化ERK1/2的大量表达。而35%O2和过氧化氢酶预处理组和35%O2预处理组,以及21% O2和30μM H2O2预处理组和21% O2预处理组磷酸化的ERK1/2表达无明显区别。4. ERK SiRNA诱导的PC12细胞效应RT-PCR显示RNA干扰后ERKmRNA表达明显减少。Western blot分析显示SP组相比NP组总的和磷酸化的ERK1/2蛋白表达量明显受到抑制。结果显示转染SiRNA的PC12细胞相比正常细胞活力明显减弱。5. Bcl-2表达Bcl-2蛋白和磷酸化的ERK蛋白在35%O2预处理组相比其它组表达明显增加,而在PD组和SiRNA组相比35%O2预处理组和21%O2预处理组表达明显下降。结论1. 35%O2预处理3小时恢复12小时能够保护低氧诱导的PC12细胞毒性。2. 35%O2预处理的PC12能够产生O2.?,而O2.?在抵抗低氧诱导的PC12细胞毒力方面发挥关键作用。3. 35%O2预处理细胞保护作用的的信号转导途径为MAPK。4. ERK的激活导致Bcl-2蛋白的过表达,Bcl-2在低氧诱导的PC12细胞毒性中发挥关键的保护作用。更多还原

【Abstract】 Reactive oxygen species, the collective term for superoxide anion (O2.-), hydrogen peroxide (H2O2), hydrol radical (.OH) and others, have been traditionally regarded as cytotoxic, with the potential to cause damage to lipids, protein, and DNA. In contrast to this harmful condition, considerable evidence show that various stimuli can stimulate the enzymatic generation of lower levels of ROS, acting as second messengers in response to these factors.Previous studies have indicated that ischemic preconditioning (IPC) can protect tissues against injury. Now, the concept has been extended to preconditioning triggered by non-ischemic stress, such as generation of reactive oxygen radicals.Extracellular signal-regulated kinase (ERK), a prototype member of the mitogen-activated protein kinase (MAPK) family, is up-regulated by reactive oxygen species production in response to numerous stimuli. Moreover, ERK activation promotes Bcl-2 gene expression through a variety of transcription factors activation. The Bcl-2 protein family has an important role in the regulation of programmed cell death.This article is designed to investigate whether 35% oxygen preconditioning for 3 hours can protect PC12 cells against the death induced by hypoxia (1% O2), and whether 35% oxygen induced adaptive cytoprotection is related to the generation of ROS, ERK signal-transduction pathway, and the overexpression of Bcl-2.MethodsPC12 cells, were maintained in Dulbecco’s modified Eagle’s medium. PC12 cells were preconditioned with exposure to 35% O2 for 180 min followed by 12 h recovery and subsequent exposure to hypoxia for 72 h. Prior to exposing to 35% O2 and 1% O2, the medium was replaced with serum-free medium. Cells were pre-incubated with related drugs for 60 min before exposing to 35% O2. Viability was measured by MTT method. Intracellular ROS generation was monitored by flow cytometry using peroxide-sensitive fluorescent probe 2’,7’-dichlorofluorescein diacetate and dihydroethidium. PC12 cells were transfected using lipofectamine 2000 and ERK SiRNA, as recommended by manufacturer’s instructions. ERK mRNA was detected by RT-PCR according to the manufacturer’s protocol. Western Blot was performed to estimate the expression of ERK and Bcl-2Results1. cell viability Hypoxia could cause considerable PC12 cells death. But the effect was inhibited by preconditioning with 35% O2 for 3 h followed by 12 h recovery and no efffect was found by 0 h or 24 h recovery, while preconditioning by 50% and 75% O2 just showed the opposite results in MTT assay.2. ROS production PC12 cells in 35% O2 and pyrogallol group produced more O2.? than that in the 21% O2 group and the O2.? in tempol group decreased significantly than that in the 35% O2 group. The production of H2O2 by 35% O2 preconditioning did not increased significantly compared with that of 21% O2 preconditioning. The cell viability increased significantly in pyrogallol and 35% O2 group, and reduced significantly in tempol group.3. ERK expression Thirty-five percent oxygen pretreatment significantly enhanced the cell viability and activated ERK1/2 expression, and these effect was obviously attenuated by PD98059, not by H7 and wortmannin. The activated ERK1/2 overexpression induced by 35% O2 was obviously reduced by treatment with 4-hydroxyl-tempol. Preconditioning with 21% O2 and pyrogallol, could also induced the overexpression of phosphorylated ERK1/2. Contrastively pretreatment with 35% O2 and catalase, or preconditioning with 21% O2 and H2O2 did not change the ERK1/2 expression obviously compared with that of 35% O2 or 21% O2 preconditioning respectively.4. ERK SiRNA RT-PCR analysis showed that ERK mRNA expression was remarkably inhibited by SiRNA. The expression of total and activated ERK1/2 protein in SP group was strongly suppressed. The viability of PC12 cells tranfected with ERK SiRNA was remarkably reduced than that of normal PC12 cells.5. Bcl-2 expression The levels of Bcl-2 and phosphorylated ERK expression increased obviously in 35% O2 group compared with that in other groups and decreased significantly in PD group and SiRNA group compared with that in 21% O2 or 35% O2 group.Conclusion1. 35% O2 preconditioning for 3 h followed by 12 h recovery could protect PC12 cells against death induced by hypoxia.2. PC12 cells preconditioned with 35% O2 could generate O2.?, which played a vital role in protecting PC12 cell against cytotoxicity induced by hypoxia.3. MAPK signal transduction pathway was involved in the cytoprotective effect of 35% O2 preconditioning.4. ERK signal pathway was necessary to the overexpression of the Bcl-2 protein in PC12 cells preconditioned with 35%O2, and Bcl-2 played an important role in this process.更多还原