發(fā)布時(shí)間：2018-03-18 18:28

  本文選題：視黃酸　切入點(diǎn)：跨上皮電阻　出處：《重慶醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：第一部分·RA促進(jìn)腸上皮細(xì)胞Caco-2之間的緊密連接 目的明確RA對(duì)Caco-2細(xì)胞緊密連接的促進(jìn)作用，篩選RA最佳作用濃度，探索RA信號(hào)通路中RARs與TLRs和緊密連接相關(guān)蛋白在RA促進(jìn)Caco-2細(xì)胞發(fā)揮屏障功能中的變化。 方法利用電壓電阻儀檢測(cè)RA處理后的Caco-2細(xì)胞跨上皮電阻（TER）的變化。給予不同RA濃度（0.5μmol/L、1μmol/L、5μmol/L、10μmol/L、20μmol/L）處理Caco-2細(xì)胞，Real-time PCR和Western blot技術(shù)檢測(cè)分析RA作用后Caco-2細(xì)胞中RARs、TLRs和緊密連接相關(guān)蛋白的表達(dá)水平變化。免疫熒光染色觀察RA作用Caco-2細(xì)胞后關(guān)鍵受體RAR的定位表達(dá)差異。 結(jié)果RA處理顯著增加Caco-2細(xì)胞跨上皮電阻，與未處理組比較具有統(tǒng)計(jì)學(xué)差異，P0.01。不同濃度RA（0.5μmol/L、1μmol/L、5μmol/L、10μmol/L、20μmol/L）分別處理Caco-2細(xì)胞后，Real-time PCR檢測(cè)結(jié)果顯示RAR、TLR4和ZO-2的mRNA表達(dá)水平均較RA未處理組有明顯升高，且RA為1-10μmol/L時(shí)效果最為顯著（P0.05，P0.001）；Western blot進(jìn)一步分析RAR、TLR4和ZO-2三個(gè)蛋白表達(dá)水平變化，其結(jié)果與real-time PCR檢測(cè)分析完全一致。但不同濃度RA對(duì)RAR、RAR和TLR2，以及Occludin、ZO-1的表達(dá)水平并沒(méi)有明顯的改變。同時(shí)，免疫熒光染色觀察到未經(jīng)RA處理的Caco-2細(xì)胞中RAR多定位于細(xì)胞核膜周邊；隨著RA作用濃度的不斷升高，發(fā)現(xiàn)RAR在胞漿中的表達(dá)逐漸增加，由核膜周邊聚集的RAR逐漸移向胞漿。 結(jié)論RA增加Caco-2細(xì)胞的跨上皮電阻，促進(jìn)其緊密連接，其機(jī)制可能是通過(guò)RAR信號(hào)通路，上調(diào)TLR4和緊密連接蛋白ZO-2的表達(dá)水平，從而實(shí)現(xiàn)RA促進(jìn)腸上皮屏障的保護(hù)作用。 第二部分RAR調(diào)節(jié)TLR4促進(jìn)Caco-2細(xì)胞緊密連接蛋白ZO-2的表達(dá) 目的利用Ad-RARβ和siRARβ重組腺病毒研究RARβ調(diào)控RA促進(jìn)Caco-2細(xì)胞緊密連接的作用，通過(guò)免疫共沉淀（Co-IP）和染色質(zhì)免疫共沉淀（ChIP）探索RARβ、TLR4和ZO-2三者之間的相互作用關(guān)系，尋找RARβ促進(jìn)ZO-2表達(dá)的具體調(diào)控機(jī)制。 方法重組Ad-RARβ和siRARβ腺病毒分別感染Caco-2細(xì)胞后，Real-time PCR和Western blot檢測(cè)分析RARβ、TLR4和ZO-2三者的表達(dá)水平變化。通過(guò)免疫共沉淀（Co-IP）技術(shù)，分別利用RARβ抗體和ZO-2抗體免疫沉淀細(xì)胞中與之結(jié)合的蛋白，Western blot檢測(cè)發(fā)生免疫共沉淀的目的蛋白。運(yùn)用染色質(zhì)免疫共沉淀（ChIP）技術(shù)，通過(guò)RARβ抗體沉淀Caco-2細(xì)胞中DNA-蛋白交聯(lián)復(fù)合物，Real-time PCR檢測(cè)分析經(jīng)RARβ抗體富集的DNA序列表達(dá)水平的差異。 結(jié)果重組腺病毒Ad-RARβ感染Caco-2細(xì)胞后，Real-time PCR和Western blot檢測(cè)結(jié)果顯示，RARβ mRNA和蛋白表達(dá)水平均有明顯升高，較RFP對(duì)照組具有顯著性的差異，P0.001；同時(shí)TLR4和ZO-2的表達(dá)水平也隨之顯著性上調(diào)；當(dāng)siRARβ感染阻斷RARβ的表達(dá)水平時(shí)，Caco-2細(xì)胞中TLR4和ZO-2的蛋白表達(dá)水平也隨之降低，，較RFP對(duì)照組相比具有統(tǒng)計(jì)學(xué)差異（P0.05，P0.01）。Co-IP檢測(cè)結(jié)果發(fā)現(xiàn)，RARβ抗體可沉淀Caco-2細(xì)胞中TLR4蛋白，ZO-2抗體可沉淀細(xì)胞中TLR4蛋白，表明在Caco-2細(xì)胞中RARβ蛋白與TLR4蛋白相互結(jié)合，TLR4蛋白與ZO-2蛋白相互結(jié)合；但RAR與ZO-2之間沒(méi)有蛋白-蛋白間的相互作用。進(jìn)一步利用ChIP技術(shù)研究表明，RARβ可與胞內(nèi)TLR4啟動(dòng)子區(qū)相結(jié)合，但與ZO-2啟動(dòng)子區(qū)不存在相互作用。 結(jié)論利用重組腺病毒分別過(guò)表達(dá)和沉默RAR，證實(shí)RA通過(guò)RAR信號(hào)通路的激活，上調(diào)TLR4和ZO-2的表達(dá)水平，從而實(shí)現(xiàn)RA促進(jìn)Caco-2細(xì)胞緊密連接的作用。其分子機(jī)制是RA核受體RARβ可結(jié)合在TLR4啟動(dòng)子區(qū)調(diào)控TLR4的表達(dá)水平，而TLR4亦可與ZO-2發(fā)生蛋白-蛋白間的相互作用，即RARβ通過(guò)調(diào)控TLR4進(jìn)而上調(diào)ZO-2的表達(dá)水平，促進(jìn)上皮屏障保護(hù)功能。
[Abstract]:The first part. RA promotes the close connection between Caco-2 of intestinal epithelial cells
Objective to clarify the role of RA in promoting the tight junction of Caco-2 cells, screen the best concentration of RA, and explore the changes of RARs and TLRs and tight junction proteins in RA signaling pathway to promote Caco-2 cells to play a barrier function in RA signaling pathway.
By using the method of detecting and processing RA voltage resistance tester of Caco-2 cells after transepithelial electrical resistance (TER) changes. Given the different concentration of RA (0.5 mol/L, 1 mol/L, 5 mol/L, 10 mol/L, 20 mol/L) treatment of Caco-2 cells, Real-time PCR and Western blot detection technology of Caco-2 cells after RA. RARs, expression of TLRs and occludin. Immunofluorescence staining to observe expression localization of RA Caco-2 cells after key receptor RAR.
Results RA treatment significantly increased Caco-2 cell transepithelial electrical resistance, compared with the untreated group with significant difference, different concentrations of P0.01. RA (0.5 mol/L, 1 mol/L, 5 mol/L, 10 mol/L, 20 mol/L) respectively after treatment of Caco-2 cells, Real-time PCR showed that RAR expression levels of TLR4 and ZO-2 mRNA were lower than RA in untreated group increased significantly, and the RA is 1-10 mol/L the most significant effect (P0.05, P0.001); Western blot RAR further analysis, the expression levels of three proteins TLR4 and ZO-2, and the results of real-time PCR analysis is exactly the same. But different concentrations of RA on RAR, RAR and TLR2 well, Occludin, the expression level of ZO-1 did not significantly change. At the same time, immunofluorescence staining was observed in RA treated Caco-2 cells in RAR are mostly located in the cell membrane surrounding; with the concentration of RA increased RAR expression in cytoplasm increased gradually Adding, the RAR gathered around the nuclear membrane gradually moved to the cytoplasm.
Conclusion RA increases the trans epithelial resistance of Caco-2 cells and promotes their tight junctions. The mechanism may be that the expression of TLR4 and tight junction protein ZO-2 is up-regulated by RAR signaling pathway, so as to achieve the protective effect of RA on intestinal epithelial barrier.
Second part RAR regulates TLR4 to promote the expression of close connexin ZO-2 in Caco-2 cells
Objective to promote the close connection of Caco-2 regulation of RA cells by Ad-RAR beta and siRAR recombinant adenovirus on RAR beta, by CO immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP) to explore RAR beta, interactions between TLR4 and ZO-2 three, the specific regulatory mechanism for promoting the expression of ZO-2 beta RAR.
Methods the recombinant adenovirus Ad-RAR beta and beta siRAR respectively in Caco-2 cells infected with Real-time PCR and Western blot analysis of RAR beta, change the expression level of TLR4 and ZO-2 three. By CO immunoprecipitation (Co-IP) technology, respectively using RAR beta and ZO-2 antibody immunoprecipitation cells with Western protein, blot detection of the target protein. Co immunoprecipitation using chromatin immunoprecipitation (ChIP) technology, the precipitation of DNA- protein cross-linked complex in Caco-2 cells by RAR beta antibody PCR detection and analysis by Real-time, the difference of DNA sequence table RAR beta antibody enriched expression.
Results the recombinant adenovirus Ad-RAR beta Caco-2 cells infected with Real-time PCR and Western blot assay showed that RAR beta mRNA and protein expression levels were significantly increased, compared with RFP control group with significant difference, P0.001; at the same time, the expression of TLR4 and ZO-2 also significantly increases; when the siRAR beta blocking expression of RAR infection beta, the expression level of TLR4 and ZO-2 protein in Caco-2 cells decreased, compared with RFP control group compared with statistical difference (P0.05, P0.01).Co-IP test results found that RAR beta TLR4 protein antibody precipitation in Caco-2 cells, ZO-2 antibody precipitation of TLR4 proteins in the cell, shows that the combination of RAR and TLR4 beta protein protein in Caco-2 cells. The combination of TLR4 and ZO-2 proteins; but no interaction between protein and protein between RAR and ZO-2. Further research by ChIP technology showed that RAR beta can TLR4 and intracellular. The promoter region is combined, but it does not interact with the ZO-2 promoter region.
Conclusion the recombinant adenovirus were overexpression and silencing of RAR, confirmed RA through activation of RAR signaling pathway, up regulate the expression of TLR4 and ZO-2, so as to realize the RA promote tight junction Caco-2 cells. The molecular mechanisms of RA nuclear receptor RAR beta can be combined in the expression level of TLR4 promoter TLR4, and TLR4 it interacts with ZO-2 protein of RAR beta expression by regulation of TLR4 and up regulation of ZO-2, promote epithelial barrier protection.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R574

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相關(guān)期刊論文 前1條

1 徐鵬輝,高杰英,陳德蕙,陳潔,曾麗玲,何樂(lè)思,陳小章;腸上皮內(nèi)淋巴細(xì)胞對(duì)腸上皮屏障功能的影響[J];軍事醫(yī)學(xué)科學(xué)院院刊;2005年04期

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