本文選題：PC12細胞 + ROS��； 參考：《第三軍醫(yī)大學》2009年博士論文

【摘要】： 前言 活性氧包括超氧陰離子(O2.-)、過氧化氫(H2O2)、羥自由基(.OH)和其它氧化物,過去認為是有細胞毒性的,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,該活性氧可作為對這些刺激反應(yīng)的第二信使。 以前認為缺血預處理(IPC)能夠保護組織免受損傷。缺血預處理已經(jīng)作為生物界的一個普遍的細胞保護機制�，F(xiàn)在預處理的概念已經(jīng)擴展到非缺血性應(yīng)急反應(yīng),如活性氧預處理。 許多刺激可導致活性氧的產(chǎn)生,從而上調(diào)細胞外信號調(diào)節(jié)激酶(ERK)的活性。而且,ERK可以通過激活一系列轉(zhuǎn)錄因子而促進B細胞淋巴瘤白血病2(Bcl-2)基因的表達。Bcl-2蛋白家族在調(diào)節(jié)程序性死亡過程中發(fā)揮重要的作用。 本文旨在研究35%氧氣預處理3小時是否能夠保護1%氧氣誘導的PC12毒性,35%氧氣預處理誘導的細胞保護作用是否和活性氧產(chǎn)生,細胞外信號激酶途徑及Bcl-2過表達有關(guān)。 方法 PC12細胞培養(yǎng)液為DMEM液。PC12細胞先用35%O2預處理180min,然后恢復12小時,最后1%O2低氧暴露72小時。在35%O2預處理前培養(yǎng)液換為無血清培養(yǎng)液。相應(yīng)藥物加入時間為35%O2預處理前60min。細胞活力用MTT法測定。細胞內(nèi)ROS檢測采用對過氧化物敏感的熒光探針DCFH-DA和HEt。PC12細胞用脂質(zhì)體2000和SiRNA按說明書操作轉(zhuǎn)染。ERKmRNA分析采用RT-PCR法。ERK、Bcl-2表達采用Western Blot。 結(jié)果 1. 35%O2預處理對低氧誘導的PC12細胞的影響1%氧氣能導致相當數(shù)量的PC12死亡。但該細胞毒性作用能被35%O2預處理3小時恢復12小時逆轉(zhuǎn)(P0.01),而隨后的0、24小時恢復無此作用。 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預處理產(chǎn)生的H2O2相比21%O2預處理組無明顯增多。細胞活力在pyrogallol和35%O2組明顯增強,而在tempol組明顯減弱。 3. ERK表達35%O2預處理可以明顯增強細胞活力和磷酸化的ERK1/2表達,這些效應(yīng)可被ERK信號通路阻止劑PD98059而非PKA/PKC和PI3k/Akt通路阻止劑H7和wortmannin明顯減弱。 35%O2誘導的磷酸化ERK1/2的過表達可被4-羥-tempol明顯減弱。用21%O2和20μMpyrogallol預處理同樣能夠誘導磷酸化ERK1/2的大量表達。而35%O2和過氧化氫酶預處理組和35%O2預處理組,以及21% O2和30μM H2O2預處理組和21% O2預處理組磷酸化的ERK1/2表達無明顯區(qū)別。 4. ERK SiRNA誘導的PC12細胞效應(yīng)RT-PCR顯示RNA干擾后ERKmRNA表達明顯減少。Western blot分析顯示SP組相比NP組總的和磷酸化的ERK1/2蛋白表達量明顯受到抑制。結(jié)果顯示轉(zhuǎn)染SiRNA的PC12細胞相比正常細胞活力明顯減弱。 5. Bcl-2表達Bcl-2蛋白和磷酸化的ERK蛋白在35%O2預處理組相比其它組表達明顯增加,而在PD組和SiRNA組相比35%O2預處理組和21%O2預處理組表達明顯下降。 結(jié)論 1. 35%O2預處理3小時恢復12小時能夠保護低氧誘導的PC12細胞毒性。 2. 35%O2預處理的PC12能夠產(chǎn)生O2.?,而O2.?在抵抗低氧誘導的PC12細胞毒力方面發(fā)揮關(guān)鍵作用。 3. 35%O2預處理細胞保護作用的的信號轉(zhuǎn)導途徑為MAPK。 4. ERK的激活導致Bcl-2蛋白的過表達,Bcl-2在低氧誘導的PC12細胞毒性中發(fā)揮關(guān)鍵的保護作用。
[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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R363

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