發(fā)布時(shí)間：2018-06-12 20:07

  本文選題：內(nèi)皮細(xì)胞 + 第三丁基過(guò)氧化氫��； 參考：《福建醫(yī)科大學(xué)》2009年碩士論文

【摘要】： 目的:研究NF-κB-iNOS-NO信號(hào)通路在第三丁基過(guò)氧化氫(Tert-butylhydroperoxide,t-BHP)誘導(dǎo)內(nèi)皮細(xì)胞損傷中的作用,在此基礎(chǔ)上進(jìn)一步研究重組脂聯(lián)素對(duì)氧化應(yīng)激狀態(tài)下內(nèi)皮細(xì)胞的NF-κB-iNOS-NO信號(hào)表達(dá)的影響。 方法:體外培養(yǎng)人臍靜脈內(nèi)皮細(xì)胞(HUVEC),用t-BHP誘導(dǎo)刺激,模擬體外內(nèi)皮細(xì)胞氧化應(yīng)激損傷的細(xì)胞模型,通過(guò)MTT法觀察t-BHP作用后細(xì)胞的存活率,Griess化學(xué)顯色法檢測(cè)氧化損傷過(guò)程中細(xì)胞內(nèi)NO水平,同時(shí)應(yīng)用Western blot技術(shù)檢測(cè)胞漿及胞核核因子κB片段p65(NF-κBp65)蛋白、誘導(dǎo)型一氧化氮合酶(inducible nitric oxide synthase,iNOS)蛋白表達(dá)水平的變化。應(yīng)用iNOS抑制劑左旋硝基精氨酸甲酯(N~G-Nitro-L-arginine Methyl Ester,L-NAME)預(yù)處理并檢測(cè)細(xì)胞內(nèi)NO水平。隨后用含脂聯(lián)素基因的重組腺病毒處理,(選MOI100,作用時(shí)間48h)觀察重組脂聯(lián)素對(duì)t-BHP誘導(dǎo)內(nèi)皮細(xì)胞損傷的保護(hù)作用。 結(jié)果:MTT顯示t-BHP(12.5-100umol/L)作用不同時(shí)間(30-120min)后可明顯抑制內(nèi)皮細(xì)胞的生長(zhǎng),并呈現(xiàn)一定的時(shí)效和量效關(guān)系,50umol/L濃度的t-BHP處理60min時(shí)胞漿iNOS蛋白達(dá)高峰水平,處理30min時(shí)胞核NF-κB活化片段p65蛋白達(dá)高峰水平,iNOS抑制劑L-NAME(600umol/L)預(yù)處理細(xì)胞24h后可明顯抑制內(nèi)皮細(xì)胞胞內(nèi)NO水平的升高。加入含脂聯(lián)素基因的重組腺病毒處理,(選MOI100,作用時(shí)間48h)干預(yù)處理后可抑制t-BHP誘導(dǎo)的內(nèi)皮細(xì)胞胞內(nèi)NO水平的升高,并且重組脂聯(lián)素能抑制t-BHP誘導(dǎo)的內(nèi)皮細(xì)胞胞核NF-κBp65及胞漿iNOS蛋白的表達(dá)水平。 結(jié)論:(1)t-BHP可抑制內(nèi)皮細(xì)胞的生長(zhǎng),在次過(guò)程中伴隨細(xì)胞內(nèi)NO水平的升高,(2)氧化應(yīng)激NF-κB-iNOS-NO信號(hào)通路可能是t-BHP誘導(dǎo)內(nèi)皮細(xì)胞發(fā)生損傷的重要通路之一。(3)重組脂聯(lián)素可以抑制t-BHP誘導(dǎo)內(nèi)皮細(xì)胞損傷,同時(shí)抑制細(xì)胞NO水平,(4)重組脂聯(lián)素可能通過(guò)抑制細(xì)胞內(nèi)NF-κB活化,胞漿iNOS表達(dá)水平來(lái)抑制NO水平,最終減輕氧化應(yīng)激誘導(dǎo)的內(nèi)皮細(xì)胞損傷。
[Abstract]:Aim: to investigate the role of NF- 魏 B-iNOS-NO signaling pathway in endothelial cell injury induced by Tert-butylhydroperoxidet-BHP (Ding Ji), and to investigate the effect of recombinant adiponectin on the expression of NF- 魏 B-iNOS-NO signal in endothelial cells under oxidative stress. Methods: human umbilical vein endothelial cells (HUVECs) were cultured in vitro and stimulated by t-BHP. MTT assay was used to observe the survival rate of the cells treated with t-BHP and the level of no in the cells during oxidative injury was detected by Griess chemical staining. Meanwhile, the cytoplasm and nuclear factor 魏 B fragment p65NF- 魏 B p65 protein were detected by Western blot. Expression of inducible nitric oxide synthase (iNOS) protein. NG-Nitro-L-arginine methyl ester (NG-Nitro-L-arginine Methyl EsterL-NAME-NAME), an iNOS inhibitor, was used to pretreat and detect the level of no in the cells. Then the protective effect of recombinant adiponectin on t-BHP induced endothelial cell injury was observed with recombinant adenovirus treated with adiponectin gene (MOI 100 for 48 h). Results after treated with t-BHPX 12.5-100 umol / L for 30 to 120 min, the growth of endothelial cells was significantly inhibited, and the expression of iNOS protein reached the peak level in 60min treated with t-BHP at 50 umoll / L concentration in a time-dependent and dose-dependent manner. After treated with 30min, the p65 activation fragment of nuclear NF- 魏 B reached the peak level. Pretreatment with L-NAME-600 umol / L (iNOS inhibitor) for 24 h significantly inhibited the increase of no level in endothelial cells. After treated with recombinant adenovirus containing adiponectin gene (MOI100 for 48h), the increase of no level in endothelial cells induced by t-BHP was inhibited. The recombinant adiponectin inhibited the expression of NF- 魏 B p65 and iNOS protein in the nucleus of endothelial cells induced by t-BHP. Conclusion the growth of endothelial cells could be inhibited by TBP. The NF- 魏 B-iNOS-NO signaling pathway of oxidative stress may be one of the important pathways of t-BHP induced endothelial cell injury. The recombinant adiponectin can inhibit the injury induced by t-BHP. The recombined adiponectin may inhibit the level of no by inhibiting the activation of NF- 魏 B and the expression of iNOS in the cytoplasm, and ultimately alleviate the injury of endothelial cells induced by oxidative stress.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R363

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本文編號(hào)：2010906