TRPV4对结肠上皮屏障功能的影响及其机制研究

发布时间：2018-04-30 16:48

本文选题：紧密连接蛋白 + 磷酸化　；参考：《安徽医科大学》2017年硕士论文

【摘要】：背景溃疡性结肠炎(Ulcerative colitis,UC)是以免疫反应和黏膜损伤为特征的肠道炎症性疾病。有研究表明,肠屏障功能损伤是UC发生发展过程中的重要因素,可导致细胞间隙通透性增加,细菌、内毒素及大分子物质容易穿过粘膜屏障诱导局部免疫功能紊乱,从而加重病理损伤。紧密连接蛋白的差异表达可导致紧密连接功能的障碍。最近研究发现在实验性结肠炎中Claudin-1、Claudin-3、Claudin-4、Claudin-5、Claudin-7和Claudin-8的表达下调。然而,这些研究都不足以证实Claudin表达的下调是导致UC产生的原因还是UC病变导致的结果。最近有文献报道,上皮细胞的屏障功能不仅与某些紧密连接蛋白的差异表达有关,其磷酸化修饰所导致的紧密连接蛋白变构、胞吞和极性改变也是影响其功能的重要因素。其中,紧密连接蛋白某些基团适度的磷酸化是维持其功能的必要条件,但异常的磷酸化会改变它们之间的结合方式,影响聚集和结构稳定性,导致跨上皮阻抗和上皮间隙通透性的改变,从而影响上皮屏障功能。国内外研究也表明,肠粘膜屏障功能与某些紧密连接蛋白的磷酸化修饰改变有关。脂多糖(Lipopolysaccharide,LPS)是免疫细胞的激活剂,能够导致急性肺损伤,同时伴随着Claudin-4的表达下调。有文献报道,在胆管上皮细胞单层中,LPS引起Claudin-1和Claudin-4的再分布。目前,很少有文献报道在结肠上皮细胞中LPS对Claudin磷酸化的影响。在本研究中,我们探讨了在葡聚糖硫酸钠(Dextran Sulfate Sodium 5000,DSS)诱导的结肠炎大鼠结肠中Claudin磷酸化状态的改变;同时评估了结肠形态学指标、黏膜通透性和血清C-反应蛋白(C-Reactive protein,CRP)的改变。此外,我们还检测了LPS对T84细胞中Claudin及其磷酸化水平的影响。目的1.探讨在DSS诱导的大鼠结肠炎中结肠Claudin的磷酸化状态的改变。2.探讨LPS对结肠上皮细胞中Claudin磷酸化的影响。方法1.制备结肠炎模型实验采用健康雌性SD大鼠,随机分为正常对照组(6只)和DSS结肠炎模型组(18只)。正常组饮用蒸馏水7天作为对照,DSS结肠炎模型组自由饮用4%DSS水,于造模的第5、7、9天,分段收取大鼠的标本,观察相关指标。2.酶联免疫吸附测定(Enzyme Linked Immunosorbent Assay,ELISA)利用ELISA法检测实验大鼠血清中CRP水平。3.免疫组织化学染色利用免疫组化方法分别检测磷酸化Claudin-3、磷酸化Claudin-5、磷酸化Claudin-6、磷酸化Claudin-7在实验大鼠结肠上皮中的定位与表达。4.蛋白免疫印迹用Western Blotting检测实验大鼠结肠上皮中磷酸化Claudin的表达水平;在细胞水平,用Western Blotting检测了LPS对T84细胞中Claudin磷酸化水平的影响。结果1.DSS诱导的结肠炎模型大鼠结肠损伤程度明显高于正常对照组;且随着DSS刺激时间的延长,损伤程度加重,黏膜通透性逐渐增加。2.炎症因子CRP随着DSS刺激的延长而呈现出先高后低的规律。3.磷酸化Claudin-3和磷酸化Claudin-6免疫组化染色分布在结肠上皮隐窝的侧面和腺管腔中。磷酸化Claudin-5免疫组化染色主要分布在肠上皮隐窝的顶端、侧面和腺管腔。磷酸化Claudin-7分布在结肠上皮隐窝的顶端和腺管腔。4.磷酸化Claudin-4和磷酸化Claudin-7的表达水平在DSS实验性结肠炎的第6天和第8天增加,磷酸化Claudin-6的表达水平在第4天和第8天增加。磷酸化Claudin-5的表达水平在第4天减少,却在第8天增加。5.与对照组相比,LPS干预T84细胞48 h后,磷酸化Claudin-3的表达水平明显增加;72小时后,与对照组相比,磷酸化Claudin-3的表达水平明显降低,磷酸化Claudin-6表达增加,磷酸化Claudin-5表达降低,与体内研究结果相符。结论1.DSS诱导的结肠炎模型大鼠随着病情进展,结肠黏膜通透性逐渐增加,Claudin-4、Claudin-6、Claudin-7磷酸化水平升高,Claudin-5磷酸化水平呈现先高后低的趋势;2.LPS干预结肠上皮细胞引起Claudin磷酸化水平升高,长时间干预可诱导Claudin磷酸化水平下降。背景溃疡性结肠炎(ulcerative colitis,UC)是结肠慢性非特异性炎症,临床上主要表现为反复发作的粘液脓血便和腹痛腹泻,发病机制不明,病理损伤主要位于粘膜层与粘膜下层。结肠上皮的屏障功能障碍是导致炎症发生及加重的重要因素,其中,结肠上皮细胞间紧密连接是维系肠道屏障功能的重要结构,组成蛋白Claudin某些基团的适度磷酸化是维持其功能的必要条件,异常的磷酸化会改变Claudin间的结合方式,影响紧密连接的聚集和结构稳定性,进而导致跨上皮阻抗和上皮通透性的改变,最终影响上皮屏障功能。TRPV4是瞬时受体电位超家族(transient receptor potential vanilloid,TRPV)中重要成员之一,是非选择性通透钙离子通道,常被认为是细胞的感受器,易被多种刺激因素激活。TRPV4在胃肠道中表达广泛,其功能改变可影响上皮屏障功能。因此,本研究观察TRPV4在结肠炎模型结肠组织中表达水平的改变,检测TRPV4对Claudin磷酸化及细胞膜定位的影响,初步探讨TRPV4对结肠上皮屏障功能的作用及机制。目的1.观察TRPV4在结肠炎模型结肠中的表达情况,检测TRPV4在上皮细胞中的功能;2.观察TRPV4与Claudin的相互作用,TRPV4对Claudin-7磷酸化及细胞膜定位的影响,初步探讨TRPV4通道对结肠上皮屏障功能的作用及其机制。方法1.结肠炎模型的制备雌性小鼠,C57BL/6小鼠,随机分四组:正常组自由饮用蒸馏水7天作为对照,DSS诱导结肠炎模型组自由饮用4%DSS水7天。药物干预组,在自由饮用4%DSS水第3天,分别使用灌胃针灌服TRPV4激动剂(GSK1016790A,50 nmol/L,1 ml/10 g)与TRPV4拮抗剂(HC067047,10μmol/L,1 ml/10 g),每日一次,于造模的第8天,收取小鼠的标本,观察相关指标。2.免疫组织化学染色利用免疫组化方法分别检测TRPV4、Claudin-7、磷酸化Claudin-7在实验动物结肠上皮中定位与表达。3.蛋白免疫印迹用Western Blotting分别检测TRPV4激动剂/拮抗剂作用于NCM460细胞后Claudin-7磷酸化水平的变化以及实验性结肠炎小鼠结肠组织中TRPV4表达水平的变化。4.免疫共沉淀使用TRPV4抗体共沉淀Claudin,检测结肠上皮细胞中TRPV4与Claudin间相互作用。5.稳转野生型Claudin-7质粒用野生型Claudin-7质粒转染NCM460细胞,筛选出稳转细胞,使用共聚焦显微镜观察并记录TRPV4激动剂和拮抗剂预处理后对细胞膜上Claudin-7表达定位的影响。6.细胞内钙离子浓度测量分别使用TRPV4通道激动剂/拮抗剂干预NCM460细胞,利用钙成像荧光显微镜系统检测细胞内钙离子浓度的变化。7.利用FD4检测结肠通透性检测实验动物血清中FD4含量,反映结肠上皮屏障功能的损伤程度。8.细胞划痕实验使用细胞划痕实验检测TRPV4激动剂和拮抗剂作用后,结肠上皮细胞修复功能的变化。结果1.与对照相比,结肠炎模型组及TRPV4拮抗剂干预的结肠炎组小鼠结肠上皮组织中TRPV4表达水平降低,TRPV4激动剂干预组结肠中TRPV4表达水平较结肠炎模型组明显升高。2.结肠上皮细胞中TRPV4和Claudin-4、Claudin-5、Claudin-7、Claudin-8存在相互作用。3.激动TRPV4通道降低结肠上皮细胞Claudin-7磷酸化水平,拮抗TRPV4通道可增强结肠上皮细胞Claudin-7磷酸化水平。4.激动TRPV4可促使Claudin-7在细胞膜上的聚集,拮抗TRPV4预处理可减少Claudin-7在细胞膜上的聚集。5.激动TRPV4明显增加NCM460细胞钙离子内流,拮抗剂预处理NCM460细胞可明显减弱激动剂的作用。6.与对照相比,结肠炎模型组及TRPV4拮抗剂干预的结肠炎组小鼠黏膜通透性明显升高,TRPV4激动剂干预可明显降低结肠炎小鼠结肠上皮通透性,TRPV4激动剂可延缓结肠炎模型血便的出现。7.细胞划痕实验发现拮抗TRPV4可明显减弱结肠上皮细胞的修复功能。结论1.激动TRPV4通道可降低实验性结肠炎模型的结肠上皮屏障损伤程度;2.结肠上皮中TRPV4-Claudin-7形成钙信号复合物,可调节Claudin-7磷酸化水平,影响Claudin-7在细胞膜上的聚集能力,改变结肠上皮细胞间的通透性,从而影响结肠上皮屏障功能。
[Abstract]:Background ulcerative colitis (Ulcerative colitis, UC) is an inflammatory disease characterized by immune response and mucosal damage. Studies have shown that the damage of intestinal barrier function is an important factor in the development of UC, which can lead to increased intercellular permeability, and bacteria, endotoxin and macromolecules are easy to pass through the mucosal barrier. Claudin-1, Claudin-3, Claudin-4, Claudin-5, Claudin-7 and Claudin-8 are down regulated in experimental colitis. However, these studies are not sufficient to confirm the downregulation of Claudin expression. The cause of the production of UC is also the result of UC disease. Recently, it has been reported that the barrier function of epithelial cells is not only related to the differential expression of some close connexin, but also an important factor affecting the function of the compact connexin caused by its phosphorylation, and the change of the endocytosis and polarity is also an important factor affecting its function. Moderate phosphorylation of some groups is a necessary condition for the maintenance of its function, but abnormal phosphorylation will change the mode of binding between them, influence aggregation and structural stability, lead to changes in epithelial impedance and interepithelial gap permeability, and thus affect the function of epithelial barrier. Lipopolysaccharide (LPS) is an activator of the immune cells. It can lead to acute lung injury, which is associated with the downregulation of Claudin-4 expression. It is reported that LPS causes the redistribution of Claudin-1 and Claudin-4 in the monolayer of bile duct epithelial cells. The effect of LPS on the phosphorylation of Claudin in the epithelial cells. In this study, we explored the changes in the Claudin phosphorylation status in the colon of rats with Dextran Sulfate Sodium 5000 (DSS) induced colitis, and also evaluated the morphological indexes, mucosal permeability and serum C- reactive protein (C-Reactive protein, CRP) of the colon. In addition, we also examined the effect of LPS on the level of Claudin and its phosphorylation in T84 cells. Objective 1. to explore the changes in the phosphorylation of Claudin in the colon of the rat colitis induced by DSS and.2. to explore the effect of LPS on the phosphorylation of Claudin in the colon epithelial cells. Methods 1. healthy female SD rats were used in the preparation of colitis model experiments. Randomly divided into the normal control group (6) and the DSS colitis model group (18 rats). The normal group drank the distilled water for 7 days as the control, and the DSS colitis model group drank the 4%DSS water freely. In the 5,7,9 day of the model, the specimens of the rats were collected in segments, and the related indexes were observed by.2. enzyme linked immunosorbent assay (Enzyme Linked Immunosorbent Assay, ELISA) using ELISA. CRP level.3. immunohistochemical staining in the serum of experimental rats was detected by immunohistochemical method to detect phosphorylated Claudin-3, phosphorylated Claudin-5, phosphorylated Claudin-6, phosphorylated Claudin-7 in the colon epithelium of experimental rats and the expression of.4. protein immunoblotting with Western Blotting detection experimental rat colon epithelium The expression level of phosphorylated Claudin; at the cell level, the effect of LPS on the level of Claudin phosphorylation in T84 cells was detected by Western Blotting. Results the colon injury degree of 1.DSS induced colitis model rats was significantly higher than that of the normal control group; and with the prolongation of DSS stimulation time, the degree of damage was aggravated, and the mucosal permeability gradually increased.2. inflammation. With the prolongation of DSS stimulation, CRP showed a high and later low regularity of.3. phosphorylation Claudin-3 and phosphorylated Claudin-6 immunohistochemical staining in the lateral and glandular cavity of the colonic epithelial recess. The phosphorylated Claudin-5 immunohistochemical staining was mainly distributed at the top of the intestinal fossa, the side and the gland lumen. Phosphorylated Claudin-7 points. The expression level of.4. phosphorylated Claudin-4 and phosphorylated Claudin-7 increased at the sixth and eighth days of DSS experimental colitis, and the expression level of phosphorylated Claudin-6 increased in fourth days and eighth days. The expression level of phosphorylated Claudin-5 decreased in fourth days, but increased in the.5. and control group at eighth days. The expression level of phosphorylated Claudin-3 was significantly increased after LPS intervention in T84 cells 48 h. After 72 hours, the expression level of phosphorylated Claudin-3 was significantly lower than that of the control group, the expression of phosphorylated Claudin-6 increased, and the expression of phosphorylated Claudin-5 decreased, which was consistent with the results of the study in vivo. Conclusion 1.DSS induced colitis model rats were associated with the condition of the disease. Progresses, the permeability of colon mucosa increased gradually, the level of phosphorylation of Claudin-4, Claudin-6, Claudin-7 increased, and the level of phosphorylation of Claudin-5 showed a trend of high and low; 2.LPS increased the level of Claudin phosphorylation in colonic epithelial cells, and prolonged the decrease in the level of phosphorylation of Claudin. Background ulcerative colitis (ulcerative CO) Litis, UC) is a chronic nonspecific inflammation of the colon. The main clinical manifestations are repeated episodes of mucus purulent stool and abdominal pain and diarrhea. The pathogenesis is unknown. The pathological damage is mainly located in the mucosa and submucosa. The barrier dysfunction of the colon epithelium is an important factor leading to the occurrence and aggravation of inflammation. Among them, the colonic epithelial cells are closely connected. It is an important structure to maintain the intestinal barrier function. The moderate phosphorylation of some group of protein Claudin is a necessary condition to maintain its function. Abnormal phosphorylation will change the binding mode of Claudin, affect the aggregation and structural stability of tight junction, and lead to the change of the upper skin impedance and epithelial permeability, and the final effect. The skin barrier function.TRPV4 is one of the important members of the transient receptor potential vanilloid (TRPV). It is a non selective permeable calcium channel. It is often considered to be a cell receptor, which is easily activated by a variety of stimulant factors and is widely used in the gastrointestinal tract. The function changes can affect the function of the epithelial barrier. This study observed the changes in the expression level of TRPV4 in colitis model colon tissue, detected the effect of TRPV4 on the phosphorylation of Claudin and the location of cell membrane, and preliminarily explored the effect and mechanism of TRPV4 on the barrier function of colon epithelium. Objective 1. to observe the expression of TRPV4 in the colon of colitis model and to detect the work of TRPV4 in the epithelial cells. 2. to observe the interaction between TRPV4 and Claudin, the effect of TRPV4 on the phosphorylation of Claudin-7 and the location of cell membrane, and preliminarily discuss the effect of TRPV4 channel on the barrier function of the colon epithelium and its mechanism. Methods the female mice and C57BL/6 mice were randomly divided into four groups: the free drinking distilled water in the normal group for 7 days as the control, and the DSS lure. The model group was free to drink 4%DSS water for 7 days. In the drug intervention group, the TRPV4 agonists (GSK1016790A, 50 nmol/L, 1 ml/10 g) and TRPV4 antagonists (HC067047,10 micron mol/L, 1 ml/10 g) were administered with a gavage for third days free drinking water for third days. Histochemical staining was used to detect TRPV4, Claudin-7, phosphorylated Claudin-7 in the colon epithelium of the experimental animals and the expression of.3. protein in the colon epithelium. The changes of Claudin-7 phosphorylation level after the action of TRPV4 agonist / antagonist on NCM460 cells and the mice of experimental colitis with Western Blotting were detected by Western Blotting. Changes in the expression level of TRPV4 in intestinal tissue.4. immunoprecipitation using TRPV4 antibody co precipitation Claudin, detection of the interaction between TRPV4 and Claudin in colon epithelial cells,.5. stabilized wild type Claudin-7 plasmids transfected to NCM460 cells with wild type Claudin-7 plasmids and screened out the stable cells, and observed and recorded TRPV4 agitation by confocal microscopy. Effect of pretreatment on the expression of Claudin-7 on cell membrane after pretreatment with agents and antagonists the intracellular calcium concentration in.6. cells was measured by TRPV4 channel agonist / antagonist, and NCM460 cells were interfered with TRPV4 channel agonist / antagonist respectively. The changes of intracellular calcium concentration were detected by calcium imaging fluorescence microscopy system,.7. was detected by FD4 for detection of colonic permeability in experimental animal serum Middle FD4 content, reflecting the damage degree of colon epithelial barrier function.8. cell scratch test using cell scratch test to detect the changes of colon epithelial cell repair function after the action of TRPV4 agonists and antagonists. Results 1. compared with control, colitis model group and TRPV4 antagonist dry pretreated colitis mice colon epithelial tissue TRPV4 The expression level of TRPV4 in the colon of TRPV4 agonist intervention group was significantly higher than that in the colitis model group. TRPV4 and Claudin-4, Claudin-5, Claudin-7, and Claudin-8 were interacted with.3. excited TRPV4 channel to reduce the Claudin-7 phosphorylation level of colon epithelial cells, and the antagonistic TRPV4 channel could enhance the colon epithelium. The Claudin-7 phosphorylation level.4. stimulated TRPV4 to stimulate the aggregation of Claudin-7 on the cell membrane. Antagonistic TRPV4 pretreatment could reduce the aggregation of Claudin-7 on the cell membrane and significantly increase the NCM460 cell calcium influx. The antagonist pretreated NCM460 cells could significantly weaken the effect of the irritable agent,.6. compared with the control, the colitis model The mucosal permeability in the colitis group of the group and the TRPV4 antagonist intervened significantly. The intervention of TRPV4 agonist could obviously reduce the permeability of colonic epithelium in the colitis mice. The TRPV4 agonist could delay the appearance of the colitis model blood and the.7. cell scratch test found that the antagonistic TRPV4 could obviously weaken the repair function of the colon epithelial cells. Conclusion 1. TRPV4 channel can reduce the degree of colonic barrier damage in the experimental colitis model. 2. the TRPV4-Claudin-7 formation of calcium signal complex in the colon epithelium can regulate the level of Claudin-7 phosphorylation, affect the aggregation of Claudin-7 on the cell membrane, change the permeability between the epithelial cells of the colon, and thus influence the function of the colon epithelial barrier.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R574.62

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