P物质调控肥大细胞脱颗粒的一种新的机制的研究

发布时间：2018-01-11 11:34

本文关键词：P物质调控肥大细胞脱颗粒的一种新的机制的研究　出处：《山西医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:利用体外诱导小鼠骨髓细胞获得的高纯度肥大细胞,探讨IL-4参与诱导获得的肥大细胞NK-1R和FcεRⅠα的表达情况;建立神经肽P物质介导的肥大细胞脱颗粒模型,初步探讨P物质除通过其特异受体NK-1R调控肥大细胞脱颗粒外是否还存在FcεRⅠα介导的途径。方法:1.从Balb/c小鼠股骨提取骨髓细胞,分不同浓度IL-4诱导组即100ng/ml干细胞因子(stem cell factor,SCF)、15ng/ml IL-3以及0,10,15,20,25ng/ml IL-4进行体外培养。每周换液,将悬浮细胞移入新的培养体系。倒置显微镜观察并记录细胞生长情况。2.四周后收集各组细胞及上清液,进行甲苯胺蓝染色、流式细胞术检测CD117鉴定肥大细胞,流式细胞术以及Western Blotting分析不同组骨髓肥大细胞FcεRⅠα和NK-1R表达情况。3.经鉴定培养成功的骨髓肥大细胞中分别加入不同浓度P物质(0、0.01、0.1、1、10μg/ml)孵育半小时,检测上清液和细胞内组胺含量,计算组胺释放率。结果:1.不同浓度IL-4诱导组(100ng/ml SCF、15ng/ml IL-3以及0,10,15,20,25ng/ml IL-4)均可诱导获得肥大细胞。2.其中SCF、IL-3和IL-4共同诱导组获得的肥大细胞FcεRⅠα和NK-1R的表达高于单纯SCF和IL-3组(0 ng/ml IL-4诱导组);而20ng/ml IL-4诱导组肥大细胞表面FcεRⅠα和NK-1R表达达最佳状态。3.20ng/ml IL-4诱导组肥大细胞可被低浓度P物质(0.01μg/ml)激活进而发生脱颗粒,其组胺释放率与SP浓度呈正相关;0 ng/ml IL-4诱导组肥大细胞表达FcεRⅠα但是几乎不表达NK-1R,此时在高浓度SP(1μg/ml)作用时肥大细胞仍可产生应答。结论:初步验证P物质调控肥大细胞脱颗粒存在其特异受体NK-1R介导的非免疫性以及FcεRⅠα介导的免疫性双途径,为下一步研究肥大细胞脱颗粒机制奠定基础,从而为变应性及非变应性炎性疾病的诊治提供新思路。
[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.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R765

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