35%氧预处理对低氧诱发PC12细胞死亡的保护作用

发布时间：2018-07-12 11:19

本文选题：PC12细胞 + ROS　；参考：《第三军医大学》2009年博士论文

【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(P0.01),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。
[Abstract]:Preface
Active oxygen, including superoxide anion (O2.-), hydrogen peroxide (H2O2), hydroxyl radical (.OH) and other oxides, was considered to be cytotoxic in the past, and was able to damage the harmful effects of lipids, proteins and DNA., and a considerable amount of evidence suggested that many stimuli could stimulate the production of small amount of active oxygen, which could be used as a stimulus to these stimuli. The second messenger of the reaction.
It was previously thought that ischemic preconditioning (IPC) could protect the tissue from injury. Ischemic preconditioning has been used as a universal cell protection mechanism in the biological community. The concept of preconditioning has now been extended to non ischemic response, such as active oxygen preconditioning.
Many stimuli can lead to the production of reactive oxygen species and up regulate the activity of extracellular signal regulated kinase (ERK). Moreover, ERK can promote the expression of B cell lymphoma leukemia 2 (Bcl-2) gene by activating a series of transcription factors. The.Bcl-2 protein family plays an important role in regulating the process of programmed death.
The purpose of this study is to investigate whether 35% oxygen preconditioning can protect the PC12 toxicity induced by oxygen for 3 hours. 35% oxygen preconditioning induced cell protection is related to the production of reactive oxygen species, the extracellular signal kinase pathway and the overexpression of Bcl-2.
Method
The PC12 cell culture solution was DMEM liquid.PC12 cells pretreated with 35%O2 for 180min, then recovered for 12 hours, and then exposed to 1%O2 hypoxic for 72 hours. Before 35%O2 pretreatment, the culture liquid was changed into a serum-free culture solution. The activity of the corresponding drug was determined by MTT method before 35%O2 pretreatment. The ROS detection in the cells was sensitive to peroxide. Fluorescent probes DCFH-DA and HEt.PC12 cells were transfected with liposomes 2000 and SiRNA according to instructions..ERKmRNA was analyzed by RT-PCR method.ERK and Bcl-2 Blot. Western Blot..
Result
The effect of 1. 35%O2 pretreatment on hypoxia induced PC12 cells 1% oxygen could lead to a considerable number of PC12 deaths. However, the cytotoxic effect of this cell could be reversed for 12 hours by 35%O2 preconditioning for 3 hours (P0.01), and the subsequent recovery of 0,24 had no effect.
2. ROS produced more PC12 than the 21%O2 group in the group of 35%O2 and pyrogallol, and the O2. produced in the Tempol group was significantly less than the H2O2 of the 35%O2 group.35%O2 pretreatment.
3. ERK expression 35%O2 preconditioning can significantly enhance cell viability and phosphorylation of ERK1/2 expression, which can be significantly reduced by ERK signaling blocking agent PD98059, not PKA/PKC and PI3k/Akt pathway inhibitor H7 and wortmannin.
The overexpression of phosphorylated ERK1/2 induced by 35%O2 can be significantly weakened by 4- hydroxy-tempol. 21%O2 and 20 micron Mpyrogallol pretreatment can also induce a large number of phosphorylated ERK1/2 expressions. The phosphorylation of 35%O2 and catalase preconditioning and 35%O2 preconditioning, as well as the phosphorylation of the 21% O2 and 30 micron M H2O2 preconditioning groups and 21% pretreatment groups There is no obvious difference.
The PC12 cell effect induced by 4. ERK SiRNA showed that the expression of ERKmRNA significantly decreased after RNA interference and the.Western blot analysis showed that the total and phosphorylation of ERK1/2 protein expression in the NP group was significantly suppressed in the SP group compared with the NP group. The results showed that the cells transfected with SiRNA were significantly less active than normal cells.
The expression of Bcl-2 protein and phosphorylated ERK protein in 5. Bcl-2 was significantly increased in the 35%O2 preconditioning group, but in the PD group and the SiRNA group, the expression of the 35%O2 preconditioning group and the 21%O2 preconditioning group decreased significantly.
conclusion
1. 35%O2 pretreatment for 3 hours to restore 12 hours can protect PC12 cytotoxicity induced by hypoxia.
2. 35%O2 pretreated PC12 can produce O2.? O2. plays a key role in resisting hypoxia induced PC12 cell toxicity.
The signal transduction pathway of 3. 35%O2 pretreatment was MAPK.
4. ERK activation leads to over expression of Bcl-2 protein, and Bcl-2 plays a key protective role in hypoxia induced PC12 cytotoxicity.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R363

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