本文選題：羥氯喹 + 腸炎相關(guān)腸癌�。� 參考：《浙江大學(xué)》2017年博士論文

【摘要】：背景:炎癥性腸病(IBD)患者,包括克羅恩氏病(CD)和潰瘍性結(jié)腸炎(UC)患者,較健康人群更易發(fā)生腸癌,由IBD發(fā)展而來的腸癌稱為腸炎相關(guān)結(jié)直腸癌(CAC)。IBD患者腸上皮屏障破損,腸道微生物進(jìn)入腸粘膜固有層,激活固有免疫細(xì)胞Toll樣受體(TLRs)炎性通路,如巨噬細(xì)胞TLR4炎性通路,釋放大量促炎因子和DNA突變誘導(dǎo)劑活性氧(ROS),誘導(dǎo)腸上皮細(xì)胞惡性變,從而導(dǎo)致腸癌發(fā)生發(fā)展。腸道炎性微環(huán)境是CAC發(fā)生的重要誘因,抑制腸道炎癥是防治CAC的重要手段。氯喹(CQ)及其衍生物羥氯喹(HCQ)除治療瘧疾外,也用于治療炎癥性疾病,如細(xì)菌感染、系統(tǒng)性紅斑狼瘡、風(fēng)濕性關(guān)節(jié)炎等。CQ/HCQ通過抑制胞內(nèi)細(xì)菌生長、促炎因子釋放、促炎信號通路活化發(fā)揮抗炎作用。此外,CQ/HCQ也能用于治療腫瘤,現(xiàn)有多項(xiàng)臨床試驗(yàn)表明:CQ/HCQ通過抑制腫瘤細(xì)胞增殖、促進(jìn)凋亡,治療非小細(xì)胞肺癌、頭頸部腫瘤和轉(zhuǎn)移性實(shí)體瘤等。有研究發(fā)現(xiàn)CQ能有效緩解輕中度活動(dòng)性UC患者腸道炎癥,但是,CQ/HCQ對IBD誘導(dǎo)而來CAC是否有防治作用未知,值得我們進(jìn)一步探討。目的:在小鼠致死性腸炎和CAC模型中,研究HCQ對腸炎和腸道腫瘤的療效,并對其抗炎和抗腫瘤機(jī)制進(jìn)行初步探討。方法:1.用高劑量葡聚糖硫酸鈉(DSS)喂予小鼠建立致死性腸炎模型,HCQ持續(xù)每天給藥,觀察小鼠存活率。2.用氧化偶氮甲烷(AOM)+DSS建立小鼠早期CAC模型,HCQ持續(xù)每天給藥,評估小鼠腸炎。分離純化小鼠腸粘膜固有層免疫細(xì)胞,Westernblot檢測炎性蛋白NF-κB、STAT3和MAPK表達(dá),實(shí)時(shí)定量PCR檢測促炎因子IL1β、IL6、TNFα、COX2、IFNβ、IP10、RANTES 和 MCP1 的 mRNA 水平,流式檢測ROS表達(dá)。3.分析基因表達(dá)綜合數(shù)據(jù)庫(GEO)微陣列數(shù)據(jù),研究TLR4基因在IBD患者和健康人群表達(dá)變化。4.體外TLR4配體脂多糖刺激巨噬細(xì)胞活化,檢測HCQ對TLR4信號通路蛋白表達(dá)、促炎因子mRNA水平和ROS生成的作用。5.用AOM+DSS建立小鼠中晚期CAC模型,HCQ持續(xù)每天給藥,觀察小鼠腸道腫瘤數(shù)目、直徑改變。分離純化小鼠腸粘膜固有層免疫細(xì)胞,Western blot檢測炎性蛋白表達(dá)。分離純化小鼠腸道腫瘤細(xì)胞,免疫組化檢測細(xì)胞增殖蛋白Ki-67、凋亡蛋白c-PARP表達(dá),Western blot檢測細(xì)胞周期調(diào)控蛋白、凋亡蛋白表達(dá),免疫熒光染色分析小鼠結(jié)腸Tunel表達(dá)。結(jié)果:1.HCQ顯著提高小鼠致死性腸炎存活率。2.HCQ緩解小鼠CAC早期腸道炎癥,具體表現(xiàn)為緩解小鼠體重下降、結(jié)腸縮短,降低疾病活動(dòng)指數(shù)和組織形態(tài)學(xué)評分。HCQ抑制腸固有層免疫細(xì)胞炎性蛋白 NK-κB、STAT3 和 MAPK 磷酸化、促炎因子 IL1 β、IL6、TNFα、COX2、IFNβ、IP10、RANTES 和 MCP1 表達(dá)、ROS 釋放。3.與健康人群相比,IBD患者腸道高表達(dá)TLR4基因。體外實(shí)驗(yàn),HCQ抑制巨噬細(xì)胞TLR4信號通路,具體表現(xiàn)為抑制TLR4通路相關(guān)蛋白活化,抑制促炎因子表達(dá)和ROS釋放。4.HCQ抑制小鼠CAC中晚期腸道腫瘤發(fā)生發(fā)展,具體表現(xiàn)為緩解小鼠體重下降,小鼠腸道腫瘤數(shù)目減少、直徑減小,降低小鼠腺癌比例。HCQ抑制腸固有層免疫細(xì)胞炎性蛋白NK-κB、STAT3和MAPK磷酸化。HCQ抑制腸癌細(xì)胞增殖、誘導(dǎo)周期停滯和細(xì)胞凋亡。結(jié)論:本課題中,我們發(fā)現(xiàn)HCQ緩解小鼠CAC模型早期腸炎和中晚期腫瘤發(fā)生發(fā)展。在小鼠CAC早期腸炎,HCQ抑制腸固有層免疫細(xì)胞炎性通路活化、促炎因子生成和DNA突變誘導(dǎo)劑ROS釋放。HCQ發(fā)揮抗炎作用可能部分是通過抑制巨噬細(xì)胞TLR4通路活化起作用的。因此,HCQ可以抑制腸炎,進(jìn)而抑制腸上皮細(xì)胞惡性變,抑制腫瘤發(fā)生發(fā)展。此外,HCQ也能直接調(diào)控腫瘤細(xì)胞。在小鼠中晚期CAC,HCQ能抑制腸道腫瘤細(xì)胞增殖、促進(jìn)其周期停滯和凋亡。總之,HCQ通過抗炎和抗腫瘤雙重作用,對小鼠CAC起防治作用。臨床上,HCQ可作為潛在藥物,防止IBD向CAC發(fā)生發(fā)展。
[Abstract]:Background: Patients with inflammatory bowel disease (IBD), including Krohn S's disease (CD) and ulcerative colitis (UC), are more likely to have colorectal cancer than in healthy people. Colorectal cancer, developed by IBD, is called the intestinal epithelial barrier of patients with colorectal cancer associated colorectal cancer (CAC).IBD, the intestinal microflora enters the lamina propria, and activates the Toll like receptor of the inherent immune cells (TL). Rs) inflammatory pathways, such as macrophage TLR4 inflammatory pathway, release a large number of proinflammatory factors and DNA mutation inducer active oxygen (ROS), induce malignant transformation of intestinal epithelial cells and lead to the development of colon cancer. Intestinal inflammatory microenvironment is an important inducement of CAC, and the inhibition of intestinal inflammation is an important means to prevent and control CAC. Chloroquine (CQ) and its derivative hydroxychloroquine (HCQ) in addition to the treatment of malaria, it is also used to treat inflammatory diseases such as bacterial infection, systemic lupus erythematosus, rheumatoid arthritis and other.CQ/HCQ by inhibiting the growth of intracellular bacteria, releasing proinflammatory cytokines, activating the inflammatory signaling pathway and activating the anti-inflammatory effects. In addition, CQ/HCQ can also be used to treat tumors. A number of clinical trials have shown that CQ/HCQ is suppressed by inhibition. The proliferation of tumor cells, the promotion of apoptosis, the treatment of non small cell lung cancer, head and neck tumor and metastatic solid tumor. Some studies have found that CQ can effectively alleviate the intestinal inflammation in patients with mild and moderate active UC, but the effect of CQ/HCQ on IBD induced CAC is unknown, and it is worth our further study. Objective: in murine fatal enteritis and CAC In the model, the effect of HCQ on enteritis and intestinal tumor was studied and its anti-inflammatory and anti-tumor mechanism was preliminarily discussed. Methods: 1. the mice were fed with high dose sodium dextran sulfate (DSS) to establish a lethal enteritis model. HCQ was administered continuously every day, and the survival rate of mice was observed by oxygen azo methane (AOM) +DSS to establish a mouse early CAC model, HCQ holding. Continued daily administration, evaluation of mice enteritis. Isolation and purification of mouse intestinal mucosa propria, Westernblot detection of inflammatory protein NF- kappa B, STAT3 and MAPK expression. Real-time quantitative PCR detection of proinflammatory cytokines IL1 beta, IL6, TNF alpha, COX2, IFN beta, IP10, and the level of expression analysis gene expression database Microarray data, the expression of TLR4 gene in IBD patients and healthy people was studied..4. in vitro TLR4 ligand lipopolysaccharide stimulated macrophage activation, the expression of HCQ on TLR4 signaling protein, the level of proinflammatory factor mRNA and the effect of ROS formation in.5. using AOM+DSS to establish middle and late CAC mold in mice. HCQ continued daily administration and observed intestinal tumor in mice. Number, diameter change. Isolation and purification of mouse intestinal mucosa propria immune cells, Western blot detection of inflammatory protein expression, isolation and purification of mouse intestinal tumor cells, immunohistochemical detection of cell proliferation protein Ki-67, apoptosis protein c-PARP expression, Western blot detection of cell cycle regulated protein, apoptosis protein expression, immunofluorescence staining analysis small Tunel expression in rat colon. Results: 1.HCQ significantly improved the survival rate of fatal enteritis in mice.2.HCQ relieving the early intestinal inflammation in CAC mice. The specific expression was to alleviate the loss of body weight, shorten the colon, reduce the disease activity index and the histomorphology score,.HCQ inhibited the intestinal lamina propria immune fine cytoinflammatory protein, NK- kappa B, STAT3 and MAPK phosphorylation, and promoted the inhibition of intestinal propria. The expression of IL1 beta, IL6, TNF alpha, COX2, IFN beta, IP10, RANTES and MCP1. ROS release.3. is high expression of TLR4 genes in the intestinal tract of IBD patients compared with healthy people. The occurrence and development of late intestinal tumor is manifested as reducing the weight loss of mice, reducing the number of intestinal tumor in mice, decreasing the diameter of intestinal cancer, reducing the proportion of adenocarcinoma in mice and inhibiting the inflammatory protein NK- kappa B of the intestinal lamina propria, STAT3 and MAPK phosphorylated.HCQ to inhibit the proliferation of colon cancer cells, induce the stagnation of cell cycle and apoptosis. We found that HCQ alleviates early enteritis and advanced tumor development in mouse CAC model. In CAC early enteritis in mice, HCQ inhibits the activation of inflammatory pathway in the intestinal lamina propria, proinflammatory factor generation and DNA mutation inducer ROS release.HCQ to play an anti-inflammatory role partly by inhibiting the activation of macrophage TLR4 pathway. Therefore, H CQ can inhibit enteritis and inhibit the malignant transformation of intestinal epithelial cells and inhibit the development of tumor. In addition, HCQ can also directly regulate tumor cells. In the middle and late stages of mouse CAC, HCQ can inhibit the proliferation of intestinal tumor cells and promote its periodic stagnation and apoptosis. In short, HCQ plays a preventive and preventive effect on mouse CAC through the double effects of anti-inflammatory and anti-tumor. HCQ can be used as a potential drug to prevent IBD from developing to CAC.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R735.34;R574

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