超低分子量肝素对神经小胶质细胞炎症反应的作用及其机制研究

发布时间：2018-01-15 00:19

本文关键词：超低分子量肝素对神经小胶质细胞炎症反应的作用及其机制研究　出处：《山东大学》2014年硕士论文　论文类型：学位论文

【摘要】：研究背景近年来的研究表明,炎症与多种神经退行性疾病的发生和发展密切相关。小胶质细胞属于脑固有免疫细胞,一旦被激活,通过释放致炎因子和活性氧自由基等分子介导神经炎症。在炎症发展的过程中,活化的小胶质细胞合成、释放多种免疫因子和细胞毒性因子,协同作用于周边胶质细胞和神经元,形成神经炎症-胶质细胞激活反馈系统,逐步放大彼此毒性,促成了慢性炎症的形成和炎性因子水平的持续升高,最终导致神经元的损伤变性甚至死亡。肝素具有抗凝、抗炎、抗过敏作用。对疾病模型的研究发现,肝素可以减轻脑缺血再灌注损伤等多种疾病模型的体内炎症反应。超低分子量肝素是肝素降解得到的寡糖片段,平均分子量为2.2kDa,与肝素相比,有较好的血脑屏障透过能力。另外,超低分子量肝素导致出血副作用的发生率较低,安全系数较高,对神经退行性病变具有潜在的治疗学价值。本课题研究超低分子量肝素对神经小胶质细胞炎症反应的影响及作用机制。实验方法抗炎作用研究：本课题以脂多糖刺激小鼠BV2小胶质细胞建立小胶质细胞炎症反应模型,MTT法评价超低分子量肝素(ULMWH)对BV2细胞活力的影响；采用硝酸还原酶法检测ULMWH对上清中NO释放的影响；ELISA法检测ULMWH对上清中炎症因子IL-1β、TNF-α释放的影响；Real-time PCR法检测ULMWH对IL-1β、TNF-α和ICAM-1mRNA表达的影响；Western blotting法检测ULMWH对iNOS等相关蛋白表达水平的影响；分别用流式细胞术和荧光显微镜观察方法检测ULMWH对细胞内ROS水平的影响。抗炎作用机制研究：细胞免疫荧光法观察ULMWH对BV2小胶质细胞中NFκB p65亚基核转位情况的影响；Western blotting法检测ULMWH对BV2小胶质细胞中PI3K/Akt/NFκB信号通路相关蛋白(p-Akt, Akt, p-NFκB,NFκB)及MAPKs信号通路相关蛋白(p-JNK/JNK, p-ERK/ERK, p-p38/p38)表达水平的影响。实验结果抗炎作用研究：MTT实验结果显示,ULMWH对BV2细胞没有细胞毒作用。硝酸还原酶法结果显示,50μg/mL的ULMWH可显著降低上清中NO的释放量。ELISA实验结果显示,25μg/mL、50μg/mL的ULMWH均可显著降低上清中TNF-α的释放量。Rea-time PCR结果显示,25μg/mL、50μg/mL的ULMWH均可显著降低BV2细胞中IL-1p和ICAM-1mRNA的表达,仅有50μg/mL的ULMWH可显著降低BV2细胞中TNF-αmRNA的表达。利用荧光探针DCFH-DA进行活性氧检测,荧光显微镜观察发现,模型组细胞内绿色荧光显著增强,反映ROS水平显著提高；25μg/mL.50μg/mL的ULMWH均能明显降低细胞内荧光强度,表明ROS水平显著下降。BV2细胞孵育荧光探针DCFH-DA标记,流式细胞仪检测与上述结果一致。Western blotting结果显示与NO和PGs、ROS生成相关的iNOS、COX-2蛋白表达量明显下降。与模型组相比,25μg/mL、50μg/mL ULMWH处理组iNOS蛋白表达量分别下降了22.22%和24.44%; COX-2蛋白表达量分别下降了25.50%和38.64%。抗炎作用机制研究：细胞免疫荧光法观察p65核转位情况,结果显示,50μg/mL ULMWH组红色荧光标记的p65蛋白亚基与蓝色荧光标记的细胞核明显分离,反映LPS诱导的p65亚基的核转移情况有所改善,p65亚基转移入核量降低。Western blotting结果显示,与模型组相比,25μg/mL、50μg/mL ULMWH处理组p-Akt、NFκB表达量降低,而p-NFκB表达量升高；反映ULMWH抑制了Akt的磷酸化和p-NFκB的脱磷酸转移入核。Western blotting结果显示,与模型组相比,25μg/mL、50μg/mL ULMWH处理组p-JNK、p-ERK、p-p38表达量均降低,而JNK、ERK、p38的表达量基本不变；反映ULMWH抑制了MAPKs细胞通路中JNK、ERK、p38三条通路的蛋白磷酸化。结论超低分子量肝素(ULMWH)具有较强的体外抗BV2小胶质细胞炎症反应的活性。通过抑制Akt的磷酸化,进而抑制p-NFκB的脱磷酸转移入核以及MAPKs细胞通路中关键蛋白的磷酸化,降低COX-2. iNOS等相关酶蛋白的表达,下调TNF-α、IL-1β和ICAM-1mRNA表达,最终降低NO、ROS等炎症介质的释放水平,从而阻止LPS诱导的BV2细胞炎症反应和氧化应激进程,对神经细胞起到保护作用。ULMWH的抗炎作用对中枢神经退行性疾病具有潜在的治疗学意义。
[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.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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