本文關(guān)鍵詞：gx-50對(duì)β淀粉樣蛋白所誘導(dǎo)小膠質(zhì)細(xì)胞趨化性的影響　出處：《上海交通大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

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【摘要】：小膠質(zhì)細(xì)胞是神經(jīng)膠質(zhì)細(xì)胞的一種，約占大腦中的神經(jīng)膠質(zhì)細(xì)胞的10～20%。目前的研究認(rèn)為小膠質(zhì)細(xì)胞是常駐在神經(jīng)組織中一種免疫細(xì)胞，它能清除中樞神經(jīng)系統(tǒng)中的損壞的神經(jīng)元，斑塊及感染性物質(zhì)。在阿爾茲海默癥（Alzheimer‘s disease,AD）的發(fā)展過(guò)程中小膠質(zhì)細(xì)胞起著非常重要的作用。腦內(nèi)老年斑是AD特征性病理表現(xiàn)之一，其主要成分β-淀粉樣蛋白（β-amyloid,Aβ）。過(guò)度活化的小膠質(zhì)細(xì)胞聚集在Aβ周圍持續(xù)釋放一些炎性因子，引起神經(jīng)組織炎癥反應(yīng)和神經(jīng)元變性。因此，通過(guò)抑制過(guò)多的小膠質(zhì)細(xì)胞向Aβ遷移，可以減少小膠質(zhì)細(xì)胞的過(guò)度活化，減輕神經(jīng)炎癥和神經(jīng)元的變性。gx-50是從花椒中提取的一種活性成分，我們實(shí)驗(yàn)室的前期研究發(fā)現(xiàn)gx-50可以有效抑制LPS誘導(dǎo)巨噬細(xì)胞釋放炎性因子,并能夠抑制Aβ誘導(dǎo)的大鼠神經(jīng)元細(xì)胞的凋亡。本研究的目的是探討gx-50對(duì)Aβ所誘導(dǎo)的大鼠小膠質(zhì)細(xì)胞趨化反應(yīng)的影響。 研究方法： 我們采用CCK-8的方法檢測(cè)不同濃度的gx-50對(duì)小膠質(zhì)細(xì)胞活性的影響。采用transwell法檢測(cè)gx-50對(duì)大鼠小膠質(zhì)細(xì)胞向Aβ遷移的影響。用ELISA檢測(cè)gx-50預(yù)處理前后對(duì)Aβ誘導(dǎo)小膠質(zhì)細(xì)胞表達(dá)趨化因子CCL5和細(xì)胞因子TGF-β1的變化情況。使用Real time PCR檢測(cè)小膠質(zhì)細(xì)胞表達(dá)TGF-β1的影響。采用Western blot檢測(cè)小膠質(zhì)細(xì)胞產(chǎn)生相關(guān)信號(hào)通路中的標(biāo)志蛋白p-Smad2，Smad7的水平以及另外一種參與小膠質(zhì)細(xì)胞遷移的蛋白p-GSK-3β的表達(dá)水平。 結(jié)果： 本研究結(jié)果顯示：通過(guò)CCK-8檢測(cè)發(fā)現(xiàn)，經(jīng)ANOVA分析0.01，0.1，1，10和100μ M的gx-50并沒(méi)有顯著影響小膠質(zhì)細(xì)胞的活性，，小膠質(zhì)細(xì)胞的活性較未經(jīng)處理的相比均達(dá)到80%以上。Transwell的實(shí)驗(yàn)結(jié)果顯示：gx-50預(yù)處理可以減少向Aβ的趨化運(yùn)動(dòng)的小膠質(zhì)細(xì)胞的數(shù)目。通過(guò)對(duì)小膠質(zhì)細(xì)胞趨化因子CCL5的表達(dá)進(jìn)行ELISA檢測(cè)，經(jīng)ANOVA分析顯示，gx-50預(yù)處理顯著降低了Aβ所誘導(dǎo)的小膠質(zhì)細(xì)胞CCL5的表達(dá)量。使用ELISA和Real time PCR的方法分別在蛋白水平和基因水平上檢測(cè)了小膠質(zhì)細(xì)胞中TGF-β1的含量，經(jīng)ANOVA分析，gx-50預(yù)處理之后，TGF-β1的分泌量在基因水平和蛋白水平與Aβ處理組相比顯著升高。Western blot的結(jié)果顯示：gx-50預(yù)處理與單獨(dú)使用Aβ相比，可以增加p-Smad2和p-GSK-3β的表達(dá)，降低Smad7的表達(dá)，并且經(jīng)ANOVA分析發(fā)現(xiàn)都達(dá)到了顯著水平。我們又對(duì)小膠質(zhì)細(xì)胞表達(dá)細(xì)胞因子及信號(hào)通路關(guān)鍵分子進(jìn)行相關(guān)回歸分析，結(jié)果顯示小膠質(zhì)細(xì)胞中CCL5的表達(dá)與Smad7存在著顯著地依存關(guān)系，而p-GSK-3β的表達(dá)與p-Smad2存在著顯著地依存關(guān)系。 結(jié)論： 綜上所述，我們的研究結(jié)果證實(shí)花椒中的成分gx-50能夠減弱小膠質(zhì)細(xì)胞向Aβ的聚集；抑制了小膠質(zhì)細(xì)胞中趨化因子CCL5的表達(dá)，減輕了局部的炎癥反應(yīng)；而經(jīng)過(guò)信號(hào)通路分析發(fā)現(xiàn)，gx-50正是由于活化了TGF-β1-smad2信號(hào)通路從而抑制了CCL5的分泌；此外gx-50還可以增加GSK-3β的磷酸化從而降低它的活性從而抑制小膠質(zhì)細(xì)胞向Aβ的聚集。由此可見(jiàn)：gx-50可以通過(guò)修復(fù)Aβ對(duì)TGF-β1-smad2信號(hào)通路造成的損傷和抑制GSK-3β活性的從而有效地抑制Aβ誘導(dǎo)的小膠質(zhì)細(xì)胞趨化運(yùn)動(dòng)，防止Aβ造成的進(jìn)一步的神經(jīng)組織炎癥反應(yīng)，對(duì)阿爾茲海默癥的發(fā)病具有一定的延緩作用。
[Abstract]:Microglia are a type of glial cell, accounting for glial cells in the brain of the 10 ~ 20%. current research that microglia are resident in neural tissues for immune cells, it can eliminate the damage of central nervous system neurons, plaque and infectious substances in Alzheimer's disease (. Alzheimer 's disease, AD) in the development of small and medium-sized glial cells play a very important role. Senile plaques in the brain is one of the pathological characteristics of AD, the main component of amyloid beta (beta -amyloid and A beta). Microglial cells activation gathered in A beta around the sustained release of some inflammatory disease factor, cause nerve inflammation and neuronal degeneration. Therefore, the migration to A beta by inhibiting excessive microglia, can reduce the excessive activation of microglia, alleviate nerve inflammation and neuronal degeneration in.Gx-50 is from the pepper An active ingredient extraction, our previous study found that gx-50 can effectively inhibit the LPS induced release of inflammatory cytokines in macrophages, apoptosis and suppresses A beta induced neuronal cells of rats. The purpose of this study is to investigate the effect of gx-50 on A beta induced rat microglial cells chemotaxis.
Research methods:
We used the method of CCK-8 gx-50 detection of different concentrations on the activation of microglial cells was detected by Transwell. The influence of gx-50 on rat microglia to A beta migration. The expression of chemokine CCL5 and cytokine TGF- beta 1 beta on A induced microglial cells before and after pretreatment of ELISA detection gx-50 the use of Real time PCR. Detection of microglia affect the expression of TGF- beta 1. The detection of Western blot microglia in signal pathway related protein markers p-Smad2, Smad7 and another in microglia migration protein p-GSK-3 beta expression level.
Result錛

本文編號(hào)：1433331