RA促进肠上皮Caco-2细胞间紧密连接的机制研究

发布时间：2018-03-18 18:28

本文选题：视黄酸　切入点：跨上皮电阻　出处：《重庆医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：第一部分·RA促进肠上皮细胞Caco-2之间的紧密连接目的明确RA对Caco-2细胞紧密连接的促进作用，筛选RA最佳作用浓度，探索RA信号通路中RARs与TLRs和紧密连接相关蛋白在RA促进Caco-2细胞发挥屏障功能中的变化。方法利用电压电阻仪检测RA处理后的Caco-2细胞跨上皮电阻（TER）的变化。给予不同RA浓度（0.5μmol/L、1μmol/L、5μmol/L、10μmol/L、20μmol/L）处理Caco-2细胞，Real-time PCR和Western blot技术检测分析RA作用后Caco-2细胞中RARs、TLRs和紧密连接相关蛋白的表达水平变化。免疫荧光染色观察RA作用Caco-2细胞后关键受体RAR的定位表达差异。结果RA处理显著增加Caco-2细胞跨上皮电阻，与未处理组比较具有统计学差异，P0.01。不同浓度RA（0.5μmol/L、1μmol/L、5μmol/L、10μmol/L、20μmol/L）分别处理Caco-2细胞后，Real-time PCR检测结果显示RAR、TLR4和ZO-2的mRNA表达水平均较RA未处理组有明显升高，且RA为1-10μmol/L时效果最为显著（P0.05，P0.001）；Western blot进一步分析RAR、TLR4和ZO-2三个蛋白表达水平变化，其结果与real-time PCR检测分析完全一致。但不同浓度RA对RAR、RAR和TLR2，以及Occludin、ZO-1的表达水平并没有明显的改变。同时，免疫荧光染色观察到未经RA处理的Caco-2细胞中RAR多定位于细胞核膜周边；随着RA作用浓度的不断升高，发现RAR在胞浆中的表达逐渐增加，由核膜周边聚集的RAR逐渐移向胞浆。结论RA增加Caco-2细胞的跨上皮电阻，促进其紧密连接，其机制可能是通过RAR信号通路，上调TLR4和紧密连接蛋白ZO-2的表达水平，从而实现RA促进肠上皮屏障的保护作用。第二部分RAR调节TLR4促进Caco-2细胞紧密连接蛋白ZO-2的表达目的利用Ad-RARβ和siRARβ重组腺病毒研究RARβ调控RA促进Caco-2细胞紧密连接的作用，通过免疫共沉淀（Co-IP）和染色质免疫共沉淀（ChIP）探索RARβ、TLR4和ZO-2三者之间的相互作用关系，寻找RARβ促进ZO-2表达的具体调控机制。方法重组Ad-RARβ和siRARβ腺病毒分别感染Caco-2细胞后，Real-time PCR和Western blot检测分析RARβ、TLR4和ZO-2三者的表达水平变化。通过免疫共沉淀（Co-IP）技术，分别利用RARβ抗体和ZO-2抗体免疫沉淀细胞中与之结合的蛋白，Western blot检测发生免疫共沉淀的目的蛋白。运用染色质免疫共沉淀（ChIP）技术，通过RARβ抗体沉淀Caco-2细胞中DNA-蛋白交联复合物，Real-time PCR检测分析经RARβ抗体富集的DNA序列表达水平的差异。结果重组腺病毒Ad-RARβ感染Caco-2细胞后，Real-time PCR和Western blot检测结果显示，RARβ mRNA和蛋白表达水平均有明显升高，较RFP对照组具有显著性的差异，P0.001；同时TLR4和ZO-2的表达水平也随之显著性上调；当siRARβ感染阻断RARβ的表达水平时，Caco-2细胞中TLR4和ZO-2的蛋白表达水平也随之降低，，较RFP对照组相比具有统计学差异（P0.05，P0.01）。Co-IP检测结果发现，RARβ抗体可沉淀Caco-2细胞中TLR4蛋白，ZO-2抗体可沉淀细胞中TLR4蛋白，表明在Caco-2细胞中RARβ蛋白与TLR4蛋白相互结合，TLR4蛋白与ZO-2蛋白相互结合；但RAR与ZO-2之间没有蛋白-蛋白间的相互作用。进一步利用ChIP技术研究表明，RARβ可与胞内TLR4启动子区相结合，但与ZO-2启动子区不存在相互作用。结论利用重组腺病毒分别过表达和沉默RAR，证实RA通过RAR信号通路的激活，上调TLR4和ZO-2的表达水平，从而实现RA促进Caco-2细胞紧密连接的作用。其分子机制是RA核受体RARβ可结合在TLR4启动子区调控TLR4的表达水平，而TLR4亦可与ZO-2发生蛋白-蛋白间的相互作用，即RARβ通过调控TLR4进而上调ZO-2的表达水平，促进上皮屏障保护功能。
[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.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R574

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