牛外周血中性粒细胞孕酮受体表达及其作用研究

发布时间：2018-04-21 09:54

本文选题：孕酮 + 孕酮受体　；参考：《内蒙古民族大学》2017年硕士论文

【摘要】：孕酮(Progesterone,P)是诱发牛围产期免疫抑制的关键激素之一,其浓度的动态变化在外周血中性粒细胞(polymorphonuclear neutrophil,PMN)的分布、数量、状态以及功能改变过程中发挥重要作用,但P诱导牛外周PMN的信号机制,特别是受体介导的信号转导机制仍有待阐释。本试验以阉牛外周血PMN为研究对象,分别采用q PCR、western blot方法,探讨不同浓度P在体内注射和体外诱导条件下,P核受体、膜受体和雌激素核受体、膜受体以及免疫活性分子TLR2、TLR4、及NF-κB等在基因转录和蛋白表达水平上的变化,从而为确定牛PMN的P受体表达状况、揭示牛外周血P调节PMN免疫功能的基因组信号途径和非基因组信号途径的特征、阐释牛外周血P浓度与PMN免疫活性的内在联系提供新证据。试验结果显示:1、受体表达:(1)nPRs表达:牛PMN在基因转录和蛋白水平上表达nPRs;低浓度P刺激PRB表达,加入核受体抑制剂RU486和LPS后均表现出下调趋势;高浓度P上调PRB的表达,且RU486对PRB的表达无显著性影响,在LPS引起的炎症反应中PRB表达下调。(2)mPRs(mPRα、mPRβ、PGRMC1)表达:牛PMN在基因转录和蛋白水平上表达mPRs;低浓度P下调mPRα和PGRMC1的表达,上调m PRβ表达在2h处达到峰值。在RU486和LPS作用下以上三种膜受体表达趋势无明显变化;高浓度P上调mPRβ和PGRMC1,抑制mPRα表达且在RU486和LPS作用下抑制作用逐渐解除。(3)ERs表达:低浓度P上调ERα和GPR30,在加入RU486后均表现出显著的下调趋势。2、细胞活性信号通路的信号表达:(1)MyD88:高低浓度P均上调MyD88,且在30min后达到峰值水平,低浓度P在RU486作用下抑制MyD88表达,高浓度P时无影响。(2)TLR2、TLR4:高低浓度P均下调TLR2,上调TLR4表达,且低浓度时RU486和LPS对二者的表达影响显著,均表现出抑制作用。(3)NF-κB:高浓度P抑制NF-κB表达,在RU486和LPS作用下抑制作用解除;低浓度P在30min后解除对NF-κB表达的抑制作用,RU486和LPS对其表达均无显著影响。本试验证实了牛外周PMN在基因和蛋白水平上存在nPRs、mPRs以及E受体的表达,并起到交叉介导P的非基因组效应。孕酮是通过PR介导的基因组途径以及上调或下调其膜受体引发快速的非基因组信号传导来发挥其抗炎作用;同时某些免疫调节作用可能是通过上调ERα和GPR30而非通过PR信号所传导,但仍需要进一步的实验来证明。
[Abstract]:Progesterone (P) is one of the key hormones to induce bovine perinatal immunosuppression. The dynamic change of its concentration plays an important role in the distribution, quantity, state and function of polymorphonuclear neutrophil (PMN) in peripheral blood neutrophil. However, the signal mechanism of P-induced bovine peripheral PMN, especially the receptor-mediated signal transduction mechanism, remains to be elucidated. In this study, PMN in bovine peripheral blood was used to study the nuclear receptor, membrane receptor and estrogen nuclear receptor of different concentrations of P in vivo and in vitro induced by western blot. The changes of membrane receptor, immunoreactive molecule TLR2, TLR4 and NF- 魏 B in gene transcription and protein expression, so as to determine the expression of P receptor in bovine PMN. To reveal the characteristics of genomic signal pathway and non-genomic signal pathway of bovine peripheral blood P regulating PMN immune function, and to explain the intrinsic relationship between bovine peripheral blood P concentration and PMN immune activity. The results showed that at 1: 1, the expression of PMN was: bovine PMN expressed nPRs at gene transcription and protein level; low concentration P stimulated the expression of PRB and showed a down-regulation trend after adding nuclear receptor inhibitor RU486 and LPS, and high concentration of P upregulated the expression of PRB. There was no significant effect of RU486 on the expression of PRB. In the inflammatory response induced by LPS, the expression of PRB was down-regulated. The expression of PRB was down-regulated: bovine PMN expressed mPRsat the level of gene transcription and protein, and low concentration of P down-regulated the expression of mPR 伪 and PGRMC1, and up regulated the expression of MPR 尾 at 2h. There was no significant change in the expression trend of the above three membrane receptors under the action of RU486 and LPS. High concentration of P upregulated mPR 尾 and PGRMC1, inhibited the expression of mPR 伪 and gradually uninhibited the expression of RU486 and LPS. The low concentration of P upregulated the expression of ER 伪 and GPR30. After adding RU486, both of them showed a down-regulation trend. High and low concentration P increased MyD88, and reached the peak level after 30min. Low concentration of P inhibited the expression of MyD88 under the action of RU486, but high concentration of P had no effect on TLR2TLR4: high and low concentrations of P down-regulated TLR2, upregulated the expression of TLR4, and at low concentration RU486 and LPS had significant effects on the expression of MyD88, both of which showed inhibitory effect on NF- 魏 B: high concentration of P inhibited the expression of NF- 魏 B. The inhibition of NF- 魏 B was relieved by RU486 and LPS, and the inhibition of NF- 魏 B by low concentration of P after 30min. RU486 and LPS had no significant effect on the expression of NF- 魏 B. This study confirmed that the expression of nPRsmPRs and E receptors in bovine peripheral PMN at the gene and protein levels, and played a cross-mediated non-genomic effect of P. Progesterone plays an anti-inflammatory role through PR mediated genomic pathway and up-regulation or down-regulation of its membrane receptor triggered by rapid non-genomic signal transduction. At the same time, some immunomodulatory effects may be mediated by up-regulation of ER 伪 and GPR30 rather than PR signals, but further experiments are needed to prove them.
【学位授予单位】：内蒙古民族大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S823

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