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  本文選題：內皮細胞 + 第三丁基過氧化氫��； 參考：《福建醫(yī)科大學》2009年碩士論文

【摘要】： 目的:研究NF-κB-iNOS-NO信號通路在第三丁基過氧化氫(Tert-butylhydroperoxide,t-BHP)誘導內皮細胞損傷中的作用,在此基礎上進一步研究重組脂聯(lián)素對氧化應激狀態(tài)下內皮細胞的NF-κB-iNOS-NO信號表達的影響。 方法:體外培養(yǎng)人臍靜脈內皮細胞(HUVEC),用t-BHP誘導刺激,模擬體外內皮細胞氧化應激損傷的細胞模型,通過MTT法觀察t-BHP作用后細胞的存活率,Griess化學顯色法檢測氧化損傷過程中細胞內NO水平,同時應用Western blot技術檢測胞漿及胞核核因子κB片段p65(NF-κBp65)蛋白、誘導型一氧化氮合酶(inducible nitric oxide synthase,iNOS)蛋白表達水平的變化。應用iNOS抑制劑左旋硝基精氨酸甲酯(N~G-Nitro-L-arginine Methyl Ester,L-NAME)預處理并檢測細胞內NO水平。隨后用含脂聯(lián)素基因的重組腺病毒處理,(選MOI100,作用時間48h)觀察重組脂聯(lián)素對t-BHP誘導內皮細胞損傷的保護作用。 結果:MTT顯示t-BHP(12.5-100umol/L)作用不同時間(30-120min)后可明顯抑制內皮細胞的生長,并呈現(xiàn)一定的時效和量效關系,50umol/L濃度的t-BHP處理60min時胞漿iNOS蛋白達高峰水平,處理30min時胞核NF-κB活化片段p65蛋白達高峰水平,iNOS抑制劑L-NAME(600umol/L)預處理細胞24h后可明顯抑制內皮細胞胞內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,作用時間48h)干預處理后可抑制t-BHP誘導的內皮細胞胞內NO水平的升高,并且重組脂聯(lián)素能抑制t-BHP誘導的內皮細胞胞核NF-κBp65及胞漿iNOS蛋白的表達水平。 結論:(1)t-BHP可抑制內皮細胞的生長,在次過程中伴隨細胞內NO水平的升高,(2)氧化應激NF-κB-iNOS-NO信號通路可能是t-BHP誘導內皮細胞發(fā)生損傷的重要通路之一。(3)重組脂聯(lián)素可以抑制t-BHP誘導內皮細胞損傷,同時抑制細胞NO水平,(4)重組脂聯(lián)素可能通過抑制細胞內NF-κB活化,胞漿iNOS表達水平來抑制NO水平,最終減輕氧化應激誘導的內皮細胞損傷。
[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.
【學位授予單位】：福建醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R363

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本文編號：2010906