本文關(guān)鍵詞：P物質(zhì)調(diào)控肥大細(xì)胞脫顆粒的一種新的機(jī)制的研究　出處：《山西醫(yī)科大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 肥大細(xì)胞 NK-1受體 P物質(zhì) FcεRⅠα IL-4

【摘要】：目的:利用體外誘導(dǎo)小鼠骨髓細(xì)胞獲得的高純度肥大細(xì)胞,探討IL-4參與誘導(dǎo)獲得的肥大細(xì)胞NK-1R和FcεRⅠα的表達(dá)情況;建立神經(jīng)肽P物質(zhì)介導(dǎo)的肥大細(xì)胞脫顆粒模型,初步探討P物質(zhì)除通過其特異受體NK-1R調(diào)控肥大細(xì)胞脫顆粒外是否還存在FcεRⅠα介導(dǎo)的途徑。方法:1.從Balb/c小鼠股骨提取骨髓細(xì)胞,分不同濃度IL-4誘導(dǎo)組即100ng/ml干細(xì)胞因子(stem cell factor,SCF)、15ng/ml IL-3以及0,10,15,20,25ng/ml IL-4進(jìn)行體外培養(yǎng)。每周換液,將懸浮細(xì)胞移入新的培養(yǎng)體系。倒置顯微鏡觀察并記錄細(xì)胞生長(zhǎng)情況。2.四周后收集各組細(xì)胞及上清液,進(jìn)行甲苯胺藍(lán)染色、流式細(xì)胞術(shù)檢測(cè)CD117鑒定肥大細(xì)胞,流式細(xì)胞術(shù)以及Western Blotting分析不同組骨髓肥大細(xì)胞FcεRⅠα和NK-1R表達(dá)情況。3.經(jīng)鑒定培養(yǎng)成功的骨髓肥大細(xì)胞中分別加入不同濃度P物質(zhì)(0、0.01、0.1、1、10μg/ml)孵育半小時(shí),檢測(cè)上清液和細(xì)胞內(nèi)組胺含量,計(jì)算組胺釋放率。結(jié)果:1.不同濃度IL-4誘導(dǎo)組(100ng/ml SCF、15ng/ml IL-3以及0,10,15,20,25ng/ml IL-4)均可誘導(dǎo)獲得肥大細(xì)胞。2.其中SCF、IL-3和IL-4共同誘導(dǎo)組獲得的肥大細(xì)胞FcεRⅠα和NK-1R的表達(dá)高于單純SCF和IL-3組(0 ng/ml IL-4誘導(dǎo)組);而20ng/ml IL-4誘導(dǎo)組肥大細(xì)胞表面FcεRⅠα和NK-1R表達(dá)達(dá)最佳狀態(tài)。3.20ng/ml IL-4誘導(dǎo)組肥大細(xì)胞可被低濃度P物質(zhì)(0.01μg/ml)激活進(jìn)而發(fā)生脫顆粒,其組胺釋放率與SP濃度呈正相關(guān);0 ng/ml IL-4誘導(dǎo)組肥大細(xì)胞表達(dá)FcεRⅠα但是幾乎不表達(dá)NK-1R,此時(shí)在高濃度SP(1μg/ml)作用時(shí)肥大細(xì)胞仍可產(chǎn)生應(yīng)答。結(jié)論:初步驗(yàn)證P物質(zhì)調(diào)控肥大細(xì)胞脫顆粒存在其特異受體NK-1R介導(dǎo)的非免疫性以及FcεRⅠα介導(dǎo)的免疫性雙途徑,為下一步研究肥大細(xì)胞脫顆粒機(jī)制奠定基礎(chǔ),從而為變應(yīng)性及非變應(yīng)性炎性疾病的診治提供新思路。
[Abstract]:Purpose: high purity of mast cells in mice induced by bone marrow cells obtained by in vitro, study the expression of IL-4 in mast cells obtained by NK-1R and Fc R I alpha epsilon; establish a model of mast cell degranulation mediated by substance P, preliminary study of substance P except through its specific receptor NK-1R regulates the degranulation of mast cells is there are ways to Fc s R I alpha mediated. Methods: 1. Balb/c mice bone marrow cells extracted from femoral, different concentrations of IL-4 induced group 100ng/ml stem cell factor (stem cell, factor, SCF), 15ng/ml IL-3 and 0,10,15,20,25ng/ ml IL-4 were cultured in vitro. Every week for liquid suspension cell into the new training system inverted microscope. Observe and record the growth of cells around.2. after cells were collected and the supernatant, toluidine blue staining, detection of CD117 identification of mast cells by flow cytometry and flow cytometry to Western and Blotting analysis of different groups of bone marrow mast cell Fc epsilon R I alpha and NK-1R expression of.3. was identified by culture material of different concentrations of P were added in the success of the bone marrow mast cells (0,0.01,0.1,1,10 g/ml) were incubated for half an hour, detection of histamine content in supernatant and cells, calculate the histamine release rate. Results: 1. different concentrations of IL-4 group (100ng/ml SCF, 15ng/ml IL-3 and 0,10,15,20,25ng/ml IL-4) can be obtained by mast cell.2. with SCF, the expression of IL-3 and IL-4 induced mast cell group Fc epsilon R I alpha and NK-1R was higher than that of SCF and IL-3 group (0 ng/ml IL-4 and 20ng/ml IL-4 induced group); group induced mast cell surface Fc e R I alpha and NK-1R expression of the best state.3.20ng/ml IL-4 induced group mast cells by low concentrations of substance P (0.01 g/ml) activation and degranulation and histamine release rate was positively correlated with the SP concentration of 0 ng/ml IL; -4 group induced mast cell expression of Fc I alpha epsilon R but almost no expression of NK-1R, at high concentration of SP (1 g/ml) the role of mast cells can respond. Conclusion: the preliminary verification of P material regulation of mast cell degranulation has its specific receptor NK-1R mediated immune and non immune Fc e R 1 alpha mediated by double way, for the next step of mast cell degranulation mechanisms to lay the foundation, so as to provide new ideas for the diagnosis and treatment of allergic and non allergic inflammatory diseases.

【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R765

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