本文選題：孕酮 + 孕酮受體　； 參考：《內(nèi)蒙古民族大學(xué)》2017年碩士論文

【摘要】：孕酮(Progesterone,P)是誘發(fā)牛圍產(chǎn)期免疫抑制的關(guān)鍵激素之一,其濃度的動態(tài)變化在外周血中性粒細胞(polymorphonuclear neutrophil,PMN)的分布、數(shù)量、狀態(tài)以及功能改變過程中發(fā)揮重要作用,但P誘導(dǎo)牛外周PMN的信號機制,特別是受體介導(dǎo)的信號轉(zhuǎn)導(dǎo)機制仍有待闡釋。本試驗以閹牛外周血PMN為研究對象,分別采用q PCR、western blot方法,探討不同濃度P在體內(nèi)注射和體外誘導(dǎo)條件下,P核受體、膜受體和雌激素核受體、膜受體以及免疫活性分子TLR2、TLR4、及NF-κB等在基因轉(zhuǎn)錄和蛋白表達水平上的變化,從而為確定牛PMN的P受體表達狀況、揭示牛外周血P調(diào)節(jié)PMN免疫功能的基因組信號途徑和非基因組信號途徑的特征、闡釋牛外周血P濃度與PMN免疫活性的內(nèi)在聯(lián)系提供新證據(jù)。試驗結(jié)果顯示:1、受體表達:(1)nPRs表達:牛PMN在基因轉(zhuǎn)錄和蛋白水平上表達nPRs;低濃度P刺激PRB表達,加入核受體抑制劑RU486和LPS后均表現(xiàn)出下調(diào)趨勢;高濃度P上調(diào)PRB的表達,且RU486對PRB的表達無顯著性影響,在LPS引起的炎癥反應(yīng)中PRB表達下調(diào)。(2)mPRs(mPRα、mPRβ、PGRMC1)表達:牛PMN在基因轉(zhuǎn)錄和蛋白水平上表達mPRs;低濃度P下調(diào)mPRα和PGRMC1的表達,上調(diào)m PRβ表達在2h處達到峰值。在RU486和LPS作用下以上三種膜受體表達趨勢無明顯變化;高濃度P上調(diào)mPRβ和PGRMC1,抑制mPRα表達且在RU486和LPS作用下抑制作用逐漸解除。(3)ERs表達:低濃度P上調(diào)ERα和GPR30,在加入RU486后均表現(xiàn)出顯著的下調(diào)趨勢。2、細胞活性信號通路的信號表達:(1)MyD88:高低濃度P均上調(diào)MyD88,且在30min后達到峰值水平,低濃度P在RU486作用下抑制MyD88表達,高濃度P時無影響。(2)TLR2、TLR4:高低濃度P均下調(diào)TLR2,上調(diào)TLR4表達,且低濃度時RU486和LPS對二者的表達影響顯著,均表現(xiàn)出抑制作用。(3)NF-κB:高濃度P抑制NF-κB表達,在RU486和LPS作用下抑制作用解除;低濃度P在30min后解除對NF-κB表達的抑制作用,RU486和LPS對其表達均無顯著影響。本試驗證實了牛外周PMN在基因和蛋白水平上存在nPRs、mPRs以及E受體的表達,并起到交叉介導(dǎo)P的非基因組效應(yīng)。孕酮是通過PR介導(dǎo)的基因組途徑以及上調(diào)或下調(diào)其膜受體引發(fā)快速的非基因組信號傳導(dǎo)來發(fā)揮其抗炎作用;同時某些免疫調(diào)節(jié)作用可能是通過上調(diào)ERα和GPR30而非通過PR信號所傳導(dǎo),但仍需要進一步的實驗來證明。
[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 偽 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 偽 and gradually uninhibited the expression of RU486 and LPS. The low concentration of P upregulated the expression of ER 偽 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 偽 and GPR30 rather than PR signals, but further experiments are needed to prove them.
【學(xué)位授予單位】：內(nèi)蒙古民族大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S823

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