本文選題：GC-C + Gn��； 參考：《昆明醫(yī)科大學(xué)》2017年博士論文

【摘要】：潰瘍性結(jié)腸炎(ulcerativecolitis,UC)是一種主要累及結(jié)腸的慢性特發(fā)性腸道炎癥性疾病,是炎癥性腸病(inflammatory bowel disease,IBD)的主要類型,以反復(fù)發(fā)作的腹痛、腹瀉和粘液膿血便為常見臨床癥狀。由于其病程遷延反復(fù)和結(jié)腸癌高風(fēng)險率而嚴(yán)重影響患者生活質(zhì)量,耗費大量醫(yī)療資源。近年來中國的IBD發(fā)病率逐漸升高,尤其是UC。IBD發(fā)病機(jī)制尚未完全闡明。盡管目前隨著生物制劑如英夫利西單抗的應(yīng)用,IBD患者的治療效果得到改善,但仍有部分患者對藥物治療不敏感,病情嚴(yán)重而需行結(jié)腸切除手術(shù)。因此,研究UC的病因?qū)W,尋找一種新的特異性強(qiáng)的治療靶點是本學(xué)科亟待解決的問題。腸上皮細(xì)胞間緊密連接蛋白(tight junction proteins,TJPs)作用于腸上皮形成緊密連接的腸粘膜機(jī)械屏障。當(dāng)腸粘膜屏障遭到破壞時,腸道通透性增加,易導(dǎo)致腸道炎癥的發(fā)生。近年來研究發(fā)現(xiàn)鳥苷酸環(huán)化酶C(guanylate cyclase C,GC-C)信號轉(zhuǎn)導(dǎo)通路調(diào)節(jié)腸上皮屏障功能和腸道炎癥的發(fā)生。GC-C是一種主要表達(dá)于腸上皮細(xì)胞的跨膜受體,鳥苷蛋白(guanylin,Gn)和尿鳥苷素(uroguanylin,Ugn)是其內(nèi)源性配體,亦表達(dá)于胃腸道上皮細(xì)胞。熱穩(wěn)定性腸毒素(heat-stable enterotoxin,STa)是GC-C的外源性配體,由產(chǎn)腸毒素大腸桿菌產(chǎn)生。當(dāng)這些配體(Gn/Ugn/STa)與 GC-C 結(jié)合后細(xì)胞內(nèi)環(huán)磷酸鳥苷(cyclic guanosine monophosphate cGMP)水平增高,激活GC-C信號通路,參與調(diào)節(jié)細(xì)胞內(nèi)外正常的離子濃度和腸道水電解質(zhì)平衡。然而,目前關(guān)于GC-C信號通路在腸道炎癥損傷中的作用尚未明確。本實驗研究首先發(fā)現(xiàn)UC患者結(jié)腸粘膜組織中GC-C及其內(nèi)源性配體Gn和Ugn的表達(dá)量與健康對照人群相比均明顯降低,且隨著UC患者疾病活動度增加,GC-C、Gn和Ugn的表達(dá)下降更為顯著。提示UC患者GC-C信號通路活性降低,其活性與UC疾病活動度呈負(fù)相關(guān)。GC-C信號通路可能參與了 UC的發(fā)生與發(fā)展。在上述發(fā)現(xiàn)的基礎(chǔ)上,為了進(jìn)一步說明GC-C信號通路在腸道屏障及炎癥損傷中的作用,我們運用人腸上皮細(xì)胞Caco-2單層屏障模型進(jìn)行實驗研究,通過檢測 GC-C 信號通路關(guān)鍵因子(GC-C、Gn、Ugn 和 cGMP)、TJPs(occludin、claudin-1和ZO-1)和促炎癥因子(IL-8和TNF-α)的表達(dá),發(fā)現(xiàn)細(xì)胞在白介素1β(Interleukin-1β,IL-1β)刺激下 GC-C、Gn、Ugn、cGMP、claudin-1 和 ZO-1 的表達(dá)明顯降低,細(xì)胞活力和超氧化物歧化酶(superoxidedismutase,SOD)酶活性下降,IL-8和TNF-α的表達(dá)明顯升高。經(jīng)轉(zhuǎn)染Gn過表達(dá)載體后,細(xì)胞活力、SOD酶活性、Gn、GC-C、cGMP、claudin-1和ZO-1的表達(dá)均明顯升高,單層細(xì)胞的通透性、IL-8和TNF-α的表達(dá)明顯降低。而經(jīng)轉(zhuǎn)染GC-CshRNA干擾載體的細(xì)胞在IL-1β的刺激下,細(xì)胞活力、SOD酶活性、claudin-1和ZO-1的水平下降更明顯,單層細(xì)胞的通透性、IL-8和TNF-α水平升高更為顯著。提示GC-C配體可激活處于靜止?fàn)顟B(tài)的GC-C信號通路,活化的GC-C信號通路可減輕Caco-2細(xì)胞在IL-1β誘導(dǎo)下的屏障功能受損及炎癥性損傷。最后,我們運用右旋糖酐硫酸酯鈉(dextran sodium sulfate,DSS)誘導(dǎo)的UC小鼠模型進(jìn)行GC-C信號通路的體內(nèi)功能實驗,發(fā)現(xiàn)經(jīng)注射Gn過表達(dá)慢病毒的結(jié)腸炎小鼠精神、進(jìn)食、活動好轉(zhuǎn),體重上升,稀便及粘液血便情況減輕,腸道通透性和結(jié)腸組織炎癥性評分降低,結(jié)腸組織GC-C、Gn、Ugn、cGMP、claudin-1和ZO-1的表達(dá)均升高,結(jié)腸組織和外周血清IL-8和TNF-α水平均明顯降低。提示Gn過表達(dá)慢病毒運用于UC小鼠可減輕小鼠腸道炎癥損傷,修復(fù)受損的腸粘膜屏障。本研究結(jié)果表明UC患者腸粘膜GC-C、Gn和Ugn的表達(dá)與UC疾病活動度呈負(fù)相關(guān),GC-C配體可激活處于靜止?fàn)顟B(tài)的GC-C信號通路,活化的GC-C信號通路可減輕Caco-2細(xì)胞在IL-1β誘導(dǎo)下的屏障功能受損及炎癥性損傷,并可減輕DSS誘導(dǎo)的小鼠腸道炎癥。綜合以上研究結(jié)果,我們認(rèn)為GC-C信號通路在UC的發(fā)生與發(fā)展中發(fā)揮了重要作用。GC-C受體激動劑具有用于UC患者臨床治療的潛能,為尋求IBD新的治療策略提供重要的實驗依據(jù)。第一部分GC-C及其配體Gn、Ugn在UC患者中差異性表達(dá)分析[目的]研究不同疾病活動度的UC患者結(jié)腸粘膜組織GC-C及其內(nèi)源性配體Gn和Ugn的表達(dá)差異,探討GC-C信號通路與UC及其嚴(yán)重度的關(guān)系。[方法]收集60例UC患者病變的結(jié)腸粘膜組織和20例正常對照者正常的結(jié)腸粘膜組織,運用改良的Mayo評分系統(tǒng)對UC患者的疾病活動度進(jìn)行評分,分為輕度活動組(18例)、中度活動組(23例)和重度活動組(19例)。采用實時熒光定量PCR(qRT-PCR)和蛋白質(zhì)印跡法(Western blot)方法分別檢測腸粘膜組織GC-C、Gn和UgnmRNA和蛋白質(zhì)的表達(dá)。比較分析GC-C、Gn和Ugn的轉(zhuǎn)錄和表達(dá)水平與UC疾病嚴(yán)重程度的相關(guān)性。[結(jié)果]qRT-PCR檢測結(jié)果顯示:正常對照組和輕、中、重度UC組的結(jié)腸粘膜組織中 GC-C mRNA 相對表達(dá)量分別為(1.652±0.258)、(0.525±0.240)、(0.153±0.107)和(0.034±0.020);Gn mRNA 相對表達(dá)量分別為(1.484±0.628)、(0.243±0.063)、(0.097±0.051)和(0.056±0.014);Ugn mRNA 相對表達(dá)量分別為(1.196±0.609)、(0.334±0.058)、(0.126±0.066)和(0.082±0.023)。與正常對照組相比,UC 組 GC-C、Gn、UgnmRNA的相對表達(dá)量均明顯降低,輕、中、重度UC組間GC-C、Gn、UgnmRNA的相對表達(dá)量亦有統(tǒng)計學(xué)差異,隨著疾病活動度加重,GC-C、Gn和UgnmRNA的相對表達(dá)量均明顯降低。運用Western blot方法檢測的結(jié)果顯示:各組間腸粘膜組織GC-C、Gn、Ugn蛋白相對表達(dá)量的變化與mRNA的變化一致。[結(jié)論]UC患者腸粘膜組織中GC-C、Gn和Ugn的轉(zhuǎn)錄和表達(dá)水平下調(diào),與UC疾病活動度呈負(fù)相關(guān)。第二部分GC-C信號通路在Caco-2細(xì)胞屏障及炎癥損傷中的作用研究[目的]運用Caco-2細(xì)胞單層屏障模型研究GC-C信號通路在腸上皮屏障和炎癥性損傷中的作用。[方法]將Caco-2腸上皮細(xì)胞培養(yǎng)在Transwell小室構(gòu)建單層細(xì)胞屏障模型,檢測單層細(xì)胞的跨膜電阻確定屏障的完整性。運用IL-1β刺激Caco-2細(xì)胞模擬腸上皮炎癥性細(xì)胞,并造成細(xì)胞間緊密連接結(jié)構(gòu)的破壞。采用脂質(zhì)體法分別轉(zhuǎn)染GC-C shRNA干擾載體和Gn過表達(dá)載體至細(xì)胞。經(jīng)過不同處理后,通過測定大分子物質(zhì)FD-4通過細(xì)胞的速率檢測Caco-2單層細(xì)胞的通透性;采用CCK-8法檢測細(xì)胞活力;NBT法檢測細(xì)胞SOD酶活性;ELISA方法檢測細(xì)胞上清液IL-8、TNF-α水平;采用qRT-PCR和Western blot方法分別檢測細(xì)胞GC-C信號通路關(guān)鍵因子(GC-C、Gn、Ugn 和 cGMP)、緊密連接蛋白(occludin、claudin-1 和 ZO-1)和促炎癥因子(IL-8、TNF-α)的表達(dá)。[結(jié)果]Caco-2單層細(xì)胞在培養(yǎng)的第19天,跨膜電阻值穩(wěn)定在250 Ω· cm2以上,表明腸屏障模型構(gòu)建成功。轉(zhuǎn)染Gn過表達(dá)載體48小時后,除了 Gn的表達(dá)明顯升高外,細(xì)胞GC-C和cGMP的表達(dá)亦明顯升高,Ugn的表達(dá)無明顯變化。轉(zhuǎn)染GC-CshRNA48小時后的細(xì)胞除了 GC-C的表達(dá)明顯降低外,Gn、Ugn和cGMP的表達(dá)亦明顯降低。IL-1 β刺激細(xì)胞后,細(xì)胞活力和SOD酶活性明顯降低,GC-C、Gn、Ugn、cGMP、claudin-1、ZO-1的表達(dá)均明顯降低,單層細(xì)胞的通透性、IL-8和TNF-α的水平均明顯升高。在IL-1β刺激的細(xì)胞轉(zhuǎn)染Gn過表達(dá)載體后,與過表達(dá)陰性對照組(vector control 1)相比,細(xì)胞活力、SOD酶活性、claudin-1和ZO-1的表達(dá)都明顯升高,IL-8、TNF-α的表達(dá)和細(xì)胞通透性均明顯降低。相反地,經(jīng)轉(zhuǎn)染GC-C shRNA干擾載體的細(xì)胞與干擾陰性對照組(vector control 2)相比,在IL-1β的誘導(dǎo)下IL-8、TNF-α的表達(dá)和細(xì)胞通透性升高更為顯著,細(xì)胞活力、SOD酶活性、claudin-1和ZO-1下降更為顯著。各組間occludin的表達(dá)無明顯差異。[結(jié)論]GC-C及其配體Gn和Ugn的表達(dá)相互調(diào)節(jié),其配體可激活處于靜止?fàn)顟B(tài)的GC-C信號通路,活化的GC-C信號通路可減輕Caco-2細(xì)胞在IL-1 β誘導(dǎo)下的屏障功能受損及炎癥性損傷。第三部分GC-C信號通路在UC小鼠腸道炎癥發(fā)生中的作用研究[目的]運用葡聚糖硫酸鈉(DSS)誘導(dǎo)的UC小鼠模型研究GC-C信號通路在腸道炎癥發(fā)生中的作用。[方法]隨機(jī)將60只Bal b/c小鼠分成5個組,每組有12只小鼠:(1)正常對照組(Control);(2)結(jié)腸炎組(DSS+ NS);(3)結(jié)腸炎+美沙拉嗪治療組(DSS+Mesalamine);(4)結(jié)腸炎+Gn載體治療組(DSS+ Gn);(5)結(jié)腸炎+美沙拉嗪聯(lián)合Gn載體治療組(DSS+ Mesalamine+ Gn)。給予小鼠自由飲用3%的DSS溶液1周進(jìn)行UC模型的構(gòu)建。UC模型構(gòu)建成功后,美沙拉嗪治療組的小鼠予30mg/kg的美沙拉嗪每日灌胃1次并持續(xù)1周,Gn載體治療組通過尾靜脈注射Gn過表達(dá)慢病毒每日1次治療1周。實驗第1日起觀察記錄小鼠的進(jìn)食、活動、體重、大便性狀及血便等情況。治療結(jié)束后測量各組小鼠腸道對大分子物質(zhì)FD-4的通透性。實驗2周后處死小鼠,收集小鼠外周血清和結(jié)腸組織。通過HE染色在光學(xué)顯微鏡下觀察結(jié)腸組織炎癥損傷情況進(jìn)行組織病理學(xué)評分。采用免疫組織化學(xué)方法檢測結(jié)腸組織GC-C信號通路關(guān)鍵因子(GC-C、Gn、Ugn和cGMP)、緊密連接蛋白(occludin、claudin-1和ZO-1)和促炎癥因子(IL-8、TNF-α)的表達(dá)。采用ELISA方法進(jìn)行小鼠外周血清IL-8和TNF-α水平的檢測。[結(jié)果]各治療組小鼠精神、進(jìn)食、活動較結(jié)腸炎組小鼠好轉(zhuǎn),體重均有升高,排稀便及粘液血便情況均減輕,其中美沙拉嗪聯(lián)合Gn載體治療組小鼠的改善效果最明顯。組織病理學(xué)評分顯示,結(jié)腸炎組小鼠的結(jié)腸組織學(xué)評分明顯高于正常對照組,各治療組的評分均低于結(jié)腸炎組,其中美沙拉嗪聯(lián)合Gn載體治療組的評分最低。與正常對照組相比,結(jié)腸炎組小鼠結(jié)腸組織GC-C、Gn、Ugn和cGMP的表達(dá)均明顯降低,各治療組的表達(dá)較結(jié)腸炎組小鼠均升高,以Gn載體治療組升高最為顯著。結(jié)腸炎組小鼠腸道通透性較正常對照組均明顯升高,各治療組小鼠的腸道通透性較結(jié)腸炎組均降低,以Gn載體治療組降低最為顯著。occludin、claudin-1和ZO-1在結(jié)腸炎組小鼠結(jié)腸組織中的表達(dá)較正常對照組均明顯降低,各治療組小鼠claudin-1和ZO-1的表達(dá)都明顯升高,其中美沙拉嗪聯(lián)合Gn載體治療組升高最明顯。結(jié)腸炎組小鼠結(jié)腸組織和外周血清中IL-8和TNF-α的表達(dá)水平較正常對照組均明顯升高,各治療組的表達(dá)較結(jié)腸炎組均明顯降低,以Gn載體治療組降低最為顯著。[結(jié)論]Gn過表達(dá)慢病毒單獨運用或聯(lián)合美沙拉嗪用于UC小鼠中可減輕小鼠腸道炎癥損傷,修復(fù)小鼠受損的腸粘膜屏障功能,進(jìn)一步證明GC-C信號通路在腸道中的保護(hù)性作用。
[Abstract]:Ulcerativecolitis (UC) is a chronic and idiopathic intestinal inflammatory disease involved in the colon. It is the main type of inflammatory bowel disease (IBD), with recurrent abdominal pain, diarrhea and mucus purulent stool as a common clinical symptom. In recent years, the incidence of IBD in China has been increasing, especially the pathogenesis of UC.IBD has not been fully elucidated. Although with the application of biological agents such as inflixi monoclonal antibody, the treatment effect of IBD patients has been improved, but some patients are still insensitive to drug treatment, and the condition is strict. It is important to undergo colectomy. Therefore, the study of the etiology of UC and the search for a new and specific therapeutic target are an urgent problem in this subject. The tight junction proteins (TJPs) is a closely linked intestinal mucosal mechanical barrier in the intestinal epithelium. When the intestinal mucosal barrier is destroyed, The intestinal permeability is increased and the intestinal inflammation is easy to occur. In recent years, the study found that the guanylate cyclase C (GC-C) signal transduction pathway regulates the intestinal epithelial barrier function and the occurrence of intestinal inflammation,.GC-C is a transmembrane receptor mainly expressed in intestinal epithelial cells, guanylin, Gn, and urinary guanosine (uroguanyli) (uroguanyli). N, Ugn) is an endogenous ligand and is also expressed in gastrointestinal epithelial cells. Heat-stable enterotoxin (STa) is a exogenous ligand of GC-C, which is produced by enterotoxigenic Escherichia coli. When these ligand (Gn/Ugn/STa) and GC-C are combined with GC-C, the level of cyclic guanosine (cyclic guanosine monophosphate cGMP) increases and activates it. -C signaling pathway is involved in regulating the normal ionic concentration and the balance of water and electrolyte in the intestinal tract. However, the role of the GC-C signaling pathway in intestinal inflammation is not clear. First, we found that the expression of GC-C and its endogenous ligands, Gn and Ugn, in the colon mucosa of UC patients is compared with those of the healthy control population. The expression of GC-C, Gn and Ugn decreased more significantly with the increase of the degree of disease activity in UC patients. It was suggested that the activity of GC-C signaling pathway in UC patients decreased and the activity of the UC disease activity was negatively related to the.GC-C signaling pathway, which might be involved in the occurrence and development of UC. On the basis of the above occurrence, the GC-C signaling pathway was further explained. In the intestinal barrier and the role of inflammatory damage, we used the human intestinal epithelial cell Caco-2 monolayer barrier model for experimental study. By detecting the key factors of the GC-C signaling pathway (GC-C, Gn, Ugn and cGMP), TJPs (occludin, claudin-1 and ZO-1) and the expression of inflammatory factors (IL-8 and TNF- alpha), the cells were found in the interleukin 1 beta. IL-1 beta stimulated the expression of GC-C, Gn, Ugn, cGMP, claudin-1 and ZO-1, and the activity of cells and the activity of superoxide dismutase (superoxidedismutase, SOD) decreased, and the expression of IL-8 and TNF- alpha was significantly increased. The permeability of monolayer cells, the expression of IL-8 and TNF- alpha, decreased significantly. Cell viability, SOD enzyme activity, claudin-1 and ZO-1 levels decreased more significantly under the stimulation of IL-1 beta by transfected GC-CshRNA interfering carriers. The permeability of monolayer cells, IL-8 and TNF- alpha levels increased more significantly. The GC-C ligands were activated in a state of rest. GC-C signaling pathway, activated GC-C signaling pathway can reduce the barrier function and inflammatory damage induced by IL-1 beta induced by Caco-2 cells. Finally, we use the UC mouse model induced by sodium dextran (dextran sodium sulfate, DSS) to carry out the functional experiment of the GC-C signaling pathway in vivo, and find that the injection Gn overexpression of the lentivirus has been found. The expression of GC-C, Gn, Ugn, Ugn, cGMP, claudin-1 and ZO-1 in colonic tissues increased and the levels of IL-8 and TNF- alpha in colonic tissue and peripheral blood decreased significantly. The results of this study showed that the expression of GC-C, Gn and Ugn in the intestinal mucosa of UC patients was negatively correlated with the degree of UC disease activity. The GC-C ligand could activate the GC-C signaling pathway in the static state, and the activated GC-C signal pathway could reduce the screen of Caco-2 cells under IL-1 beta induced by UC. The impairment of the barrier function and inflammatory injury can reduce the intestinal inflammation induced by DSS in mice. Combined with the above results, we believe that the GC-C signaling pathway plays an important role in the development and development of UC, and the.GC-C receptor agonist has the potential for the clinical treatment of UC patients and provides an important experimental basis for the search for a new therapeutic strategy for IBD. The first part GC-C and its ligand Gn, Ugn in UC patients' differential expression analysis [Objective] to study the difference in the expression of GC-C and its endogenous ligand Gn and Ugn in the colon mucosa of UC patients with different degree of activity of disease, and to explore the relationship between the GC-C signal pathway and the UC and its severity. [Methods] collect 60 cases of the colon mucosa of the UC patients and 20 cases. The normal colon mucosa tissue of normal controls was divided into mild activity group (18 cases), moderate activity group (23 cases) and severe activity group (19 cases) by modified Mayo scoring system (23 cases) and severe activity group (19 cases). The GC-C in intestinal mucosa was detected by real-time fluorescence quantitative PCR (qRT-PCR) and egg white mass imprinting (Western blot). The expression of Gn and UgnmRNA and protein. The correlation between the transcriptional and expression levels of GC-C, Gn and Ugn and the severity of UC disease was compared. [results]qRT-PCR detection results showed that the relative expression of GC-C mRNA in the colon mucosa of the normal control group and the moderate, severe UC group was (1.652 + 0.258), (0.525 + 0.240), (0.153 + 0.107) and 0. (0.), respectively). The relative expression of Gn mRNA was (1.484 + 0.628), (0.243 + 0.063), (0.097 + 0.051) and (0.056 + 0.014), and the relative expression of Ugn mRNA was (1.196 + 0.609) and (0.334 + 0.058) and (0.243). Compared with the normal control group, the relative expression of GC-C, Gn and UgnmRNA in the UC group decreased obviously, light, medium, and severe UC The relative expressions of GC-C, Gn and UgnmRNA between groups were also statistically different. The relative expression of GC-C, Gn and UgnmRNA decreased with the increase of the degree of disease activity. The results of Western blot methods showed that the changes in the relative expression of GC-C, Gn, Ugn protein in the intestinal mucosa of each group were in accordance with the changes of mRNA. The transcriptional and expression levels of GC-C, Gn and Ugn in the tissues are down regulated and negatively correlated with the degree of UC disease activity. Second the role of the GC-C signaling pathway in the Caco-2 cell barrier and inflammatory damage [Objective] to study the role of GC-C signaling pathway in the intestinal barrier and inflammatory injury by the Caco-2 cell monolayer barrier model. [Methods] Caco- 2 intestinal epithelial cells were cultured in the Transwell compartment to construct a monolayer cell barrier model to detect the integrity of the barrier of transmembrane resistance in monolayer cells. IL-1 beta was used to stimulate Caco-2 cells to simulate enteric dermatitis and to cause the destruction of intercellular close connection structure. Liposome method was used to transfect GC-C shRNA interference vector and Gn over table respectively. The carrier to the cell. After different treatment, the permeability of Caco-2 monolayer cells was detected by the determination of the macromolecular substance FD-4 through the cell rate; the cell viability was detected by the CCK-8 method; the NBT method was used to detect the activity of the cell SOD enzyme; the ELISA method was used to detect the IL-8, TNF- a level of the cell supernatant, and the qRT-PCR and Western blot were used to detect the cell GC-. The key factors of C signaling pathway (GC-C, Gn, Ugn and cGMP), the expression of tight connexin (occludin, claudin-1 and ZO-1) and the proinflammatory factor (IL-8, TNF- alpha). [results]Caco-2 monolayer cells in nineteenth days of culture, the transmembrane resistance value is stable at 250 Omega cm2, indicating that the intestinal barrier model is constructed successfully. After transfection of over expression vector 48 hours, except for 48 hours. The expression of GC-C and cGMP increased obviously, and the expression of GC-C and cGMP increased obviously, and the expression of Ugn had no obvious change. The expression of Gn, Ugn and cGMP decreased obviously after GC-CshRNA48 hours after transfection, and the activity of cell and SOD enzyme were obviously reduced after the expression of Gn, Ugn and cGMP. The expression of ZO-1 was significantly reduced, the permeability of monolayer cells and the level of IL-8 and TNF- alpha were significantly increased. After transfection of IL-1 beta stimulated cells to Gn overexpressed vector, the expression of cell activity, SOD enzyme activity, claudin-1 and ZO-1 increased significantly, IL-8, TNF- alpha expression and cell permeability compared with the overexpressed negative control group (vector control 1). On the contrary, the cells of the transfected GC-C shRNA interference carrier and the negative control group (vector control 2) were compared with the IL-1 beta induced IL-8, the expression of TNF- A and the cell permeability increased more significantly. The cell viability, the activity of SOD enzyme, the claudin-1 and ZO-1 decreased more significantly. There was no significant difference in the expression of occludin between the groups. [conclusion the expression of]GC-C and its ligand Gn and Ugn regulates each other and its ligand activates the GC-C signaling pathway in the static state. The activated GC-C signaling pathway reduces the barrier function and inflammatory damage induced by IL-1 beta induced Caco-2 cells. The role of the third partial GC-C signaling pathway in the intestinal inflammation in the UC rat [Objective] A UC mouse model induced by dextran sodium sulfate (DSS) was used to study the role of GC-C signaling pathway in the occurrence of intestinal inflammation. [Methods] 60 Bal b/c mice were randomly divided into 5 groups, 12 mice in each group: (1) normal control group (Control); (2) colitis group (DSS+ NS); (3) colitis + mesalazine treatment group (DSS+Mesalamine); (4) +Gn colitis +Gn The carrier treatment group (DSS+ Gn); (5) colitis + methalazine combined with Gn carrier therapy group (DSS+ Mesalamine+ Gn). After the construction of the UC model of free drinking of 3% DSS in mice for 1 weeks construction of the.UC model of UC, the mice of methalazine treatment group were given 1 times daily for 1 weeks of methalazine to 30mg/kg, and the Gn carrier treatment group passed tail. The Gn overexpression of lentivirus was treated 1 times a day for 1 weeks. The mice's feeding, activity, weight, stool character and blood stool were observed on the first day of the experiment. After the treatment, the permeability of the large molecular substance FD-4 in the mice was measured. After 2 weeks, the mice were killed and the mice were collected and the peripheral blood and colon tissues were collected. HE was stained in the light. Histopathological scores were observed under the microscope. The key factors of GC-C signaling pathway in colon tissue (GC-C, Gn, Ugn and cGMP), the expression of tight connexin (occludin, claudin-1 and ZO-1) and the expression of pro-inflammatory factors (IL-8, TNF- alpha) were detected by immunohistochemical method. [results] the level of serum IL-8 and TNF- alpha was detected. [results] the mice in the treatment group were better than the mice in the colitis group, and the body weight was better than those in the colitis group. The dilute stool and the mucus and blood stool were all lightened. The improvement effect of the methalazine combined with the Gn carrier group was the most obvious. The score of the fabric was significantly higher than that of the normal control group. The scores of all the treatment groups were lower than those of the colitis group. The score of the methalazine combined with the Gn carrier group was the lowest. Compared with the normal control group, the expression of GC-C, Gn, Ugn and cGMP in colonic tissue in the colitis group was significantly lower than that in the colitis group. The expression of each treatment group was higher than that of the colitis group and Gn loaded mice. The intestinal permeability in the colitis group was significantly higher than that in the normal control group. The intestinal permeability of the mice in the treatment group was lower than that of the colitis group, and the most significant.Occludin in the Gn carrier group was reduced. The expression of claudin-1 and ZO-1 in the intestinal tissue of the colitis group was significantly lower than that of the normal control group. The expression levels of claudin-1 and ZO-1 in the treatment group were significantly higher than those in the treatment group, and mesalazine combined with Gn carrier group was the most significant.

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

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