本文關鍵詞：超低分子量肝素對神經小膠質細胞炎癥反應的作用及其機制研究　出處：《山東大學》2014年碩士論文　論文類型：學位論文

  更多相關文章： 炎癥 超低分子量肝素 中樞神經退行性疾病 炎癥介質

【摘要】：研究背景 近年來的研究表明,炎癥與多種神經退行性疾病的發(fā)生和發(fā)展密切相關。小膠質細胞屬于腦固有免疫細胞,一旦被激活,通過釋放致炎因子和活性氧自由基等分子介導神經炎癥。在炎癥發(fā)展的過程中,活化的小膠質細胞合成、釋放多種免疫因子和細胞毒性因子,協(xié)同作用于周邊膠質細胞和神經元,形成神經炎癥-膠質細胞激活反饋系統(tǒng),逐步放大彼此毒性,促成了慢性炎癥的形成和炎性因子水平的持續(xù)升高,最終導致神經元的損傷變性甚至死亡。 肝素具有抗凝、抗炎、抗過敏作用。對疾病模型的研究發(fā)現(xiàn),肝素可以減輕腦缺血再灌注損傷等多種疾病模型的體內炎癥反應。超低分子量肝素是肝素降解得到的寡糖片段,平均分子量為2.2kDa,與肝素相比,有較好的血腦屏障透過能力。另外,超低分子量肝素導致出血副作用的發(fā)生率較低,安全系數較高,對神經退行性病變具有潛在的治療學價值。本課題研究超低分子量肝素對神經小膠質細胞炎癥反應的影響及作用機制。 實驗方法 抗炎作用研究：本課題以脂多糖刺激小鼠BV2小膠質細胞建立小膠質細胞炎癥反應模型,MTT法評價超低分子量肝素(ULMWH)對BV2細胞活力的影響；采用硝酸還原酶法檢測ULMWH對上清中NO釋放的影響；ELISA法檢測ULMWH對上清中炎癥因子IL-1β、TNF-α釋放的影響；Real-time PCR法檢測ULMWH對IL-1β、TNF-α和ICAM-1mRNA表達的影響；Western blotting法檢測ULMWH對iNOS等相關蛋白表達水平的影響；分別用流式細胞術和熒光顯微鏡觀察方法檢測ULMWH對細胞內ROS水平的影響。 抗炎作用機制研究：細胞免疫熒光法觀察ULMWH對BV2小膠質細胞中NFκB p65亞基核轉位情況的影響；Western blotting法檢測ULMWH對BV2小膠質細胞中PI3K/Akt/NFκB信號通路相關蛋白(p-Akt, Akt, p-NFκB,NFκB)及MAPKs信號通路相關蛋白(p-JNK/JNK, p-ERK/ERK, p-p38/p38)表達水平的影響。 實驗結果 抗炎作用研究：MTT實驗結果顯示,ULMWH對BV2細胞沒有細胞毒作用。硝酸還原酶法結果顯示,50μg/mL的ULMWH可顯著降低上清中NO的釋放量。ELISA實驗結果顯示,25μg/mL、50μg/mL的ULMWH均可顯著降低上清中TNF-α的釋放量。Rea-time PCR結果顯示,25μg/mL、50μg/mL的ULMWH均可顯著降低BV2細胞中IL-1p和ICAM-1mRNA的表達,僅有50μg/mL的ULMWH可顯著降低BV2細胞中TNF-αmRNA的表達。利用熒光探針DCFH-DA進行活性氧檢測,熒光顯微鏡觀察發(fā)現(xiàn),模型組細胞內綠色熒光顯著增強,反映ROS水平顯著提高；25μg/mL.50μg/mL的ULMWH均能明顯降低細胞內熒光強度,表明ROS水平顯著下降。BV2細胞孵育熒光探針DCFH-DA標記,流式細胞儀檢測與上述結果一致。Western blotting結果顯示與NO和PGs、ROS生成相關的iNOS、COX-2蛋白表達量明顯下降。與模型組相比,25μg/mL、50μg/mL ULMWH處理組iNOS蛋白表達量分別下降了22.22%和24.44%; COX-2蛋白表達量分別下降了25.50%和38.64%。 抗炎作用機制研究：細胞免疫熒光法觀察p65核轉位情況,結果顯示,50μg/mL ULMWH組紅色熒光標記的p65蛋白亞基與藍色熒光標記的細胞核明顯分離,反映LPS誘導的p65亞基的核轉移情況有所改善,p65亞基轉移入核量降低。Western blotting結果顯示,與模型組相比,25μg/mL、50μg/mL ULMWH處理組p-Akt、NFκB表達量降低,而p-NFκB表達量升高；反映ULMWH抑制了Akt的磷酸化和p-NFκB的脫磷酸轉移入核。Western blotting結果顯示,與模型組相比,25μg/mL、50μg/mL ULMWH處理組p-JNK、p-ERK、p-p38表達量均降低,而JNK、ERK、p38的表達量基本不變；反映ULMWH抑制了MAPKs細胞通路中JNK、ERK、p38三條通路的蛋白磷酸化。 結論 超低分子量肝素(ULMWH)具有較強的體外抗BV2小膠質細胞炎癥反應的活性。通過抑制Akt的磷酸化,進而抑制p-NFκB的脫磷酸轉移入核以及MAPKs細胞通路中關鍵蛋白的磷酸化,降低COX-2. iNOS等相關酶蛋白的表達,下調TNF-α、IL-1β和ICAM-1mRNA表達,最終降低NO、ROS等炎癥介質的釋放水平,從而阻止LPS誘導的BV2細胞炎癥反應和氧化應激進程,對神經細胞起到保護作用。ULMWH的抗炎作用對中樞神經退行性疾病具有潛在的治療學意義。
[Abstract]:Research background
Recent studies show that is closely related to the occurrence and development of inflammation and other neurodegenerative disorders. Brain microglia belong to the innate immune cells, once activated, through the release of proinflammatory cytokines and reactive oxygen free radical molecules mediating nerve inflammation. In the process of inflammation in the development of the synthesis of activated microglia. The release of a variety of immune factors and cell toxicity factor, synergistic effects on peripheral neurons and glial cells, formation of nerve inflammation - activation of glial cells in the feedback system, gradually enlarge each other toxicity, contributed to the formation of chronic inflammation and inflammatory factor levels continue to rise, eventually leading to degeneration of neurons damage and even death.
Heparin anticoagulant, anti-inflammatory, anti allergic effect. Research on the disease model that heparin can reduce cerebral ischemia-reperfusion injury and other diseases model in vivo inflammation. Ultra low molecular weight heparin heparin is obtained by degradation of oligosaccharides, the average molecular weight of 2.2kDa, compared with heparin, a blood brain barrier through better. In addition, ultra low molecular weight heparin hemorrhage due to a lower incidence of side effects, higher safety factor, is the value of learning potential treatment for neurodegenerative diseases. The research of ultra low molecular weight heparin effect on microglia inflammatory reaction and mechanism.
Experimental method
Study on the anti inflammatory effect: this subject to LPS stimulation of murine BV2 microglia cells to establish the microglia inflammatory reaction model, ultra low molecular weight heparin (ULMWH) method to evaluate the MTT effect on BV2 cell viability; ULMWH by nitrate reductase assay on NO release in the supernatant; ULMWH detection method of ELISA in the supernatant of inflammatory factor IL-1 effect of beta, TNF- alpha release; Real-time PCR method for detection of ULMWH IL-1 beta, TNF- alpha effect and ICAM-1mRNA expression; Western blotting method for detection of ULMWH iNOS protein expression; were determined by flow cytometry and the effect of fluorescence microscopy method for detection of ULMWH on the intracellular level of ROS.
Study on the mechanism of anti inflammation effect of cell immunofluorescence to observe the effect of ULMWH on BV2 in microglia NF kappa B subunit p65 nuclear translocation; Western blotting detection of ULMWH related proteins of PI3K/Akt/NF B signaling pathway in BV2 microglial cells (p-Akt, Akt, p-NF kappa B, NF kappa B) and MAPKs signaling pathway related proteins (p-JNK/JNK, p-ERK/ERK, p-p38/p38) expression level.
experimental result
Study on the anti-inflammatory effect of MTT: the experimental results show that ULMWH has no cytotoxic effect on BV2 cells. The nitrate reductase assay showed that 50 g/mL ULMWH could significantly reduce the release amount of.ELISA in the supernatant of NO experimental results showed that 25 g/mL and 50 g/mL ULMWH could significantly decrease the release amount of.Rea-time in the supernatant of TNF- alpha PCR results the display, 25 g/mL, 50 g/mL ULMWH could significantly decrease the expression of IL-1p and ICAM-1mRNA in BV2 cells, only 50 g/mL ULMWH can significantly decrease the expression of BV2 cells in TNF- alpha mRNA. ROS detection by fluorescence probe DCFH-DA, fluorescence microscopy showed that green fluorescence in cells in the model group significantly enhanced, reflect the ROS level increased significantly; 25 g/mL.50 g/mL ULMWH can significantly reduce the fluorescence intensity of cells showed that ROS levels were significantly decreased in.BV2 cells incubated with DCFH-DA fluorescent probe labeling, flow cytometry and The results are consistent with NO and.Western blotting showed that PGs, ROS are related to the generation of iNOS, COX-2 protein expression decreased significantly. Compared with the model group, 25 g/mL, 50 g/mL ULMWH treatment group iNOS protein expression were decreased by 22.22% and 24.44% respectively; the expression of COX-2 protein was decreased by 25.50% and 38.64%.
Study on the mechanism of anti inflammation cells were observed by immunofluorescence p65 nuclear translocation. Results showed that p65 protein subunits and blue fluorescence labeled 50 g/mL ULMWH group of red fluorescent labeled nuclei were isolated, nuclear transfer reflect the p65 subunit of LPS induced by the improved p65 subunit into the nuclear transfer to reduce the amount of.Western blotting the results show that compared with the model group, 25 g/mL, 50 g/mL ULMWH treatment group p-Akt, the expression of NF kappa B decreased, while p-NF kappa B expression increased; reflect ULMWH inhibited Akt phosphorylation and dephosphorylation p-NF kappa B nuclear transfer into.Western blotting results showed that compared with the model group, 25 g/mL, 50 g/mL ULMWH treatment group p-JNK, p-ERK, p-p38 expression was reduced, while JNK, ERK, p38 expression was unchanged; reflect ULMWH inhibited MAPKs cell pathway in JNK, ERK, p38 three protein phosphorylation pathway.
conclusion
Ultra low molecular weight heparin (ULMWH) has strong activity in vitro against BV2 microglia inflammation. By inhibiting the phosphorylation of Akt, thereby inhibiting p-NF kappa B dephosphorylation key protein and nuclear transfer into MAPKs cell pathway phosphorylation, decreased expression of COX-2. iNOS and other enzymes related to egg white, down TNF- the expression of IL-1 and ICAM-1mRNA alpha, beta, and ultimately reduce NO, ROS level and release of inflammatory mediators, thus preventing the BV2 cell inflammation and oxidative stress induced by LPS process, the anti-inflammatory effect of the protective effect of.ULMWH has the potential treatment for neurodegenerative disease of central significance of nerve cells play.

【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R96

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