本文選題：PC12細(xì)胞 + ROS　； 參考：《第三軍醫(yī)大學(xué)》2009年博士論文

【摘要】： 前言 活性氧包括超氧陰離子(O2.-)、過(guò)氧化氫(H2O2)、羥自由基(.OH)和其它氧化物,過(guò)去認(rèn)為是有細(xì)胞毒性的,能夠損害脂質(zhì)、蛋白和DNA。除外這些有害作用外,相當(dāng)多的證據(jù)表明許多刺激因素能夠刺激產(chǎn)生小量活性氧,該活性氧可作為對(duì)這些刺激反應(yīng)的第二信使。 以前認(rèn)為缺血預(yù)處理(IPC)能夠保護(hù)組織免受損傷。缺血預(yù)處理已經(jīng)作為生物界的一個(gè)普遍的細(xì)胞保護(hù)機(jī)制�，F(xiàn)在預(yù)處理的概念已經(jīng)擴(kuò)展到非缺血性應(yīng)急反應(yīng),如活性氧預(yù)處理。 許多刺激可導(dǎo)致活性氧的產(chǎn)生,從而上調(diào)細(xì)胞外信號(hào)調(diào)節(jié)激酶(ERK)的活性。而且,ERK可以通過(guò)激活一系列轉(zhuǎn)錄因子而促進(jìn)B細(xì)胞淋巴瘤白血病2(Bcl-2)基因的表達(dá)。Bcl-2蛋白家族在調(diào)節(jié)程序性死亡過(guò)程中發(fā)揮重要的作用。 本文旨在研究35%氧氣預(yù)處理3小時(shí)是否能夠保護(hù)1%氧氣誘導(dǎo)的PC12毒性,35%氧氣預(yù)處理誘導(dǎo)的細(xì)胞保護(hù)作用是否和活性氧產(chǎn)生,細(xì)胞外信號(hào)激酶途徑及Bcl-2過(guò)表達(dá)有關(guān)。 方法 PC12細(xì)胞培養(yǎng)液為DMEM液。PC12細(xì)胞先用35%O2預(yù)處理180min,然后恢復(fù)12小時(shí),最后1%O2低氧暴露72小時(shí)。在35%O2預(yù)處理前培養(yǎng)液換為無(wú)血清培養(yǎng)液。相應(yīng)藥物加入時(shí)間為35%O2預(yù)處理前60min。細(xì)胞活力用MTT法測(cè)定。細(xì)胞內(nèi)ROS檢測(cè)采用對(duì)過(guò)氧化物敏感的熒光探針DCFH-DA和HEt。PC12細(xì)胞用脂質(zhì)體2000和SiRNA按說(shuō)明書(shū)操作轉(zhuǎn)染。ERKmRNA分析采用RT-PCR法。ERK、Bcl-2表達(dá)采用Western Blot。 結(jié)果 1. 35%O2預(yù)處理對(duì)低氧誘導(dǎo)的PC12細(xì)胞的影響1%氧氣能導(dǎo)致相當(dāng)數(shù)量的PC12死亡。但該細(xì)胞毒性作用能被35%O2預(yù)處理3小時(shí)恢復(fù)12小時(shí)逆轉(zhuǎn)(P0.01),而隨后的0、24小時(shí)恢復(fù)無(wú)此作用。 2. ROS產(chǎn)生PC12在35%O2和pyrogallol組相比21%O2組能夠產(chǎn)生更多O2.?,而tempol組產(chǎn)生的O2.?明顯少于35%O2組。35%O2預(yù)處理產(chǎn)生的H2O2相比21%O2預(yù)處理組無(wú)明顯增多。細(xì)胞活力在pyrogallol和35%O2組明顯增強(qiáng),而在tempol組明顯減弱。 3. ERK表達(dá)35%O2預(yù)處理可以明顯增強(qiáng)細(xì)胞活力和磷酸化的ERK1/2表達(dá),這些效應(yīng)可被ERK信號(hào)通路阻止劑PD98059而非PKA/PKC和PI3k/Akt通路阻止劑H7和wortmannin明顯減弱。 35%O2誘導(dǎo)的磷酸化ERK1/2的過(guò)表達(dá)可被4-羥-tempol明顯減弱。用21%O2和20μMpyrogallol預(yù)處理同樣能夠誘導(dǎo)磷酸化ERK1/2的大量表達(dá)。而35%O2和過(guò)氧化氫酶預(yù)處理組和35%O2預(yù)處理組,以及21% O2和30μM H2O2預(yù)處理組和21% O2預(yù)處理組磷酸化的ERK1/2表達(dá)無(wú)明顯區(qū)別。 4. ERK SiRNA誘導(dǎo)的PC12細(xì)胞效應(yīng)RT-PCR顯示RNA干擾后ERKmRNA表達(dá)明顯減少。Western blot分析顯示SP組相比NP組總的和磷酸化的ERK1/2蛋白表達(dá)量明顯受到抑制。結(jié)果顯示轉(zhuǎn)染SiRNA的PC12細(xì)胞相比正常細(xì)胞活力明顯減弱。 5. Bcl-2表達(dá)Bcl-2蛋白和磷酸化的ERK蛋白在35%O2預(yù)處理組相比其它組表達(dá)明顯增加,而在PD組和SiRNA組相比35%O2預(yù)處理組和21%O2預(yù)處理組表達(dá)明顯下降。 結(jié)論 1. 35%O2預(yù)處理3小時(shí)恢復(fù)12小時(shí)能夠保護(hù)低氧誘導(dǎo)的PC12細(xì)胞毒性。 2. 35%O2預(yù)處理的PC12能夠產(chǎn)生O2.?,而O2.?在抵抗低氧誘導(dǎo)的PC12細(xì)胞毒力方面發(fā)揮關(guān)鍵作用。 3. 35%O2預(yù)處理細(xì)胞保護(hù)作用的的信號(hào)轉(zhuǎn)導(dǎo)途徑為MAPK。 4. ERK的激活導(dǎo)致Bcl-2蛋白的過(guò)表達(dá),Bcl-2在低氧誘導(dǎo)的PC12細(xì)胞毒性中發(fā)揮關(guān)鍵的保護(hù)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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R363

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