本文選題：緊密連接蛋白 + 磷酸化��； 參考：《安徽醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景潰瘍性結(jié)腸炎(Ulcerative colitis,UC)是以免疫反應(yīng)和黏膜損傷為特征的腸道炎癥性疾病。有研究表明,腸屏障功能損傷是UC發(fā)生發(fā)展過程中的重要因素,可導(dǎo)致細胞間隙通透性增加,細菌、內(nèi)毒素及大分子物質(zhì)容易穿過粘膜屏障誘導(dǎo)局部免疫功能紊亂,從而加重病理損傷。緊密連接蛋白的差異表達可導(dǎo)致緊密連接功能的障礙。最近研究發(fā)現(xiàn)在實驗性結(jié)腸炎中Claudin-1、Claudin-3、Claudin-4、Claudin-5、Claudin-7和Claudin-8的表達下調(diào)。然而,這些研究都不足以證實Claudin表達的下調(diào)是導(dǎo)致UC產(chǎn)生的原因還是UC病變導(dǎo)致的結(jié)果。最近有文獻報道,上皮細胞的屏障功能不僅與某些緊密連接蛋白的差異表達有關(guān),其磷酸化修飾所導(dǎo)致的緊密連接蛋白變構(gòu)、胞吞和極性改變也是影響其功能的重要因素。其中,緊密連接蛋白某些基團適度的磷酸化是維持其功能的必要條件,但異常的磷酸化會改變它們之間的結(jié)合方式,影響聚集和結(jié)構(gòu)穩(wěn)定性,導(dǎo)致跨上皮阻抗和上皮間隙通透性的改變,從而影響上皮屏障功能。國內(nèi)外研究也表明,腸粘膜屏障功能與某些緊密連接蛋白的磷酸化修飾改變有關(guān)。脂多糖(Lipopolysaccharide,LPS)是免疫細胞的激活劑,能夠?qū)е录毙苑螕p傷,同時伴隨著Claudin-4的表達下調(diào)。有文獻報道,在膽管上皮細胞單層中,LPS引起Claudin-1和Claudin-4的再分布。目前,很少有文獻報道在結(jié)腸上皮細胞中LPS對Claudin磷酸化的影響。在本研究中,我們探討了在葡聚糖硫酸鈉(Dextran Sulfate Sodium 5000,DSS)誘導(dǎo)的結(jié)腸炎大鼠結(jié)腸中Claudin磷酸化狀態(tài)的改變;同時評估了結(jié)腸形態(tài)學(xué)指標、黏膜通透性和血清C-反應(yīng)蛋白(C-Reactive protein,CRP)的改變。此外,我們還檢測了LPS對T84細胞中Claudin及其磷酸化水平的影響。目的1.探討在DSS誘導(dǎo)的大鼠結(jié)腸炎中結(jié)腸Claudin的磷酸化狀態(tài)的改變。2.探討LPS對結(jié)腸上皮細胞中Claudin磷酸化的影響。方法1.制備結(jié)腸炎模型實驗采用健康雌性SD大鼠,隨機分為正常對照組(6只)和DSS結(jié)腸炎模型組(18只)。正常組飲用蒸餾水7天作為對照,DSS結(jié)腸炎模型組自由飲用4%DSS水,于造模的第5、7、9天,分段收取大鼠的標本,觀察相關(guān)指標。2.酶聯(lián)免疫吸附測定(Enzyme Linked Immunosorbent Assay,ELISA)利用ELISA法檢測實驗大鼠血清中CRP水平。3.免疫組織化學(xué)染色利用免疫組化方法分別檢測磷酸化Claudin-3、磷酸化Claudin-5、磷酸化Claudin-6、磷酸化Claudin-7在實驗大鼠結(jié)腸上皮中的定位與表達。4.蛋白免疫印跡用Western Blotting檢測實驗大鼠結(jié)腸上皮中磷酸化Claudin的表達水平;在細胞水平,用Western Blotting檢測了LPS對T84細胞中Claudin磷酸化水平的影響。結(jié)果1.DSS誘導(dǎo)的結(jié)腸炎模型大鼠結(jié)腸損傷程度明顯高于正常對照組;且隨著DSS刺激時間的延長,損傷程度加重,黏膜通透性逐漸增加。2.炎癥因子CRP隨著DSS刺激的延長而呈現(xiàn)出先高后低的規(guī)律。3.磷酸化Claudin-3和磷酸化Claudin-6免疫組化染色分布在結(jié)腸上皮隱窩的側(cè)面和腺管腔中。磷酸化Claudin-5免疫組化染色主要分布在腸上皮隱窩的頂端、側(cè)面和腺管腔。磷酸化Claudin-7分布在結(jié)腸上皮隱窩的頂端和腺管腔。4.磷酸化Claudin-4和磷酸化Claudin-7的表達水平在DSS實驗性結(jié)腸炎的第6天和第8天增加,磷酸化Claudin-6的表達水平在第4天和第8天增加。磷酸化Claudin-5的表達水平在第4天減少,卻在第8天增加。5.與對照組相比,LPS干預(yù)T84細胞48 h后,磷酸化Claudin-3的表達水平明顯增加;72小時后,與對照組相比,磷酸化Claudin-3的表達水平明顯降低,磷酸化Claudin-6表達增加,磷酸化Claudin-5表達降低,與體內(nèi)研究結(jié)果相符。結(jié)論1.DSS誘導(dǎo)的結(jié)腸炎模型大鼠隨著病情進展,結(jié)腸黏膜通透性逐漸增加,Claudin-4、Claudin-6、Claudin-7磷酸化水平升高,Claudin-5磷酸化水平呈現(xiàn)先高后低的趨勢;2.LPS干預(yù)結(jié)腸上皮細胞引起Claudin磷酸化水平升高,長時間干預(yù)可誘導(dǎo)Claudin磷酸化水平下降。背景潰瘍性結(jié)腸炎(ulcerative colitis,UC)是結(jié)腸慢性非特異性炎癥,臨床上主要表現(xiàn)為反復(fù)發(fā)作的粘液膿血便和腹痛腹瀉,發(fā)病機制不明,病理損傷主要位于粘膜層與粘膜下層。結(jié)腸上皮的屏障功能障礙是導(dǎo)致炎癥發(fā)生及加重的重要因素,其中,結(jié)腸上皮細胞間緊密連接是維系腸道屏障功能的重要結(jié)構(gòu),組成蛋白Claudin某些基團的適度磷酸化是維持其功能的必要條件,異常的磷酸化會改變Claudin間的結(jié)合方式,影響緊密連接的聚集和結(jié)構(gòu)穩(wěn)定性,進而導(dǎo)致跨上皮阻抗和上皮通透性的改變,最終影響上皮屏障功能。TRPV4是瞬時受體電位超家族(transient receptor potential vanilloid,TRPV)中重要成員之一,是非選擇性通透鈣離子通道,常被認為是細胞的感受器,易被多種刺激因素激活。TRPV4在胃腸道中表達廣泛,其功能改變可影響上皮屏障功能。因此,本研究觀察TRPV4在結(jié)腸炎模型結(jié)腸組織中表達水平的改變,檢測TRPV4對Claudin磷酸化及細胞膜定位的影響,初步探討TRPV4對結(jié)腸上皮屏障功能的作用及機制。目的1.觀察TRPV4在結(jié)腸炎模型結(jié)腸中的表達情況,檢測TRPV4在上皮細胞中的功能;2.觀察TRPV4與Claudin的相互作用,TRPV4對Claudin-7磷酸化及細胞膜定位的影響,初步探討TRPV4通道對結(jié)腸上皮屏障功能的作用及其機制。方法1.結(jié)腸炎模型的制備雌性小鼠,C57BL/6小鼠,隨機分四組:正常組自由飲用蒸餾水7天作為對照,DSS誘導(dǎo)結(jié)腸炎模型組自由飲用4%DSS水7天。藥物干預(yù)組,在自由飲用4%DSS水第3天,分別使用灌胃針灌服TRPV4激動劑(GSK1016790A,50 nmol/L,1 ml/10 g)與TRPV4拮抗劑(HC067047,10μmol/L,1 ml/10 g),每日一次,于造模的第8天,收取小鼠的標本,觀察相關(guān)指標。2.免疫組織化學(xué)染色利用免疫組化方法分別檢測TRPV4、Claudin-7、磷酸化Claudin-7在實驗動物結(jié)腸上皮中定位與表達。3.蛋白免疫印跡用Western Blotting分別檢測TRPV4激動劑/拮抗劑作用于NCM460細胞后Claudin-7磷酸化水平的變化以及實驗性結(jié)腸炎小鼠結(jié)腸組織中TRPV4表達水平的變化。4.免疫共沉淀使用TRPV4抗體共沉淀Claudin,檢測結(jié)腸上皮細胞中TRPV4與Claudin間相互作用。5.穩(wěn)轉(zhuǎn)野生型Claudin-7質(zhì)粒用野生型Claudin-7質(zhì)粒轉(zhuǎn)染NCM460細胞,篩選出穩(wěn)轉(zhuǎn)細胞,使用共聚焦顯微鏡觀察并記錄TRPV4激動劑和拮抗劑預(yù)處理后對細胞膜上Claudin-7表達定位的影響。6.細胞內(nèi)鈣離子濃度測量分別使用TRPV4通道激動劑/拮抗劑干預(yù)NCM460細胞,利用鈣成像熒光顯微鏡系統(tǒng)檢測細胞內(nèi)鈣離子濃度的變化。7.利用FD4檢測結(jié)腸通透性檢測實驗動物血清中FD4含量,反映結(jié)腸上皮屏障功能的損傷程度。8.細胞劃痕實驗使用細胞劃痕實驗檢測TRPV4激動劑和拮抗劑作用后,結(jié)腸上皮細胞修復(fù)功能的變化。結(jié)果1.與對照相比,結(jié)腸炎模型組及TRPV4拮抗劑干預(yù)的結(jié)腸炎組小鼠結(jié)腸上皮組織中TRPV4表達水平降低,TRPV4激動劑干預(yù)組結(jié)腸中TRPV4表達水平較結(jié)腸炎模型組明顯升高。2.結(jié)腸上皮細胞中TRPV4和Claudin-4、Claudin-5、Claudin-7、Claudin-8存在相互作用。3.激動TRPV4通道降低結(jié)腸上皮細胞Claudin-7磷酸化水平,拮抗TRPV4通道可增強結(jié)腸上皮細胞Claudin-7磷酸化水平。4.激動TRPV4可促使Claudin-7在細胞膜上的聚集,拮抗TRPV4預(yù)處理可減少Claudin-7在細胞膜上的聚集。5.激動TRPV4明顯增加NCM460細胞鈣離子內(nèi)流,拮抗劑預(yù)處理NCM460細胞可明顯減弱激動劑的作用。6.與對照相比,結(jié)腸炎模型組及TRPV4拮抗劑干預(yù)的結(jié)腸炎組小鼠黏膜通透性明顯升高,TRPV4激動劑干預(yù)可明顯降低結(jié)腸炎小鼠結(jié)腸上皮通透性,TRPV4激動劑可延緩結(jié)腸炎模型血便的出現(xiàn)。7.細胞劃痕實驗發(fā)現(xiàn)拮抗TRPV4可明顯減弱結(jié)腸上皮細胞的修復(fù)功能。結(jié)論1.激動TRPV4通道可降低實驗性結(jié)腸炎模型的結(jié)腸上皮屏障損傷程度;2.結(jié)腸上皮中TRPV4-Claudin-7形成鈣信號復(fù)合物,可調(diào)節(jié)Claudin-7磷酸化水平,影響Claudin-7在細胞膜上的聚集能力,改變結(jié)腸上皮細胞間的通透性,從而影響結(jié)腸上皮屏障功能。
[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.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R574.62

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本文編號：1825357