羟氯喹对肠炎相关结直肠癌的防治作用及机制研究

发布时间：2018-06-14 21:19

本文选题：羟氯喹 + 肠炎相关肠癌　；参考：《浙江大学》2017年博士论文

【摘要】：背景:炎症性肠病(IBD)患者,包括克罗恩氏病(CD)和溃疡性结肠炎(UC)患者,较健康人群更易发生肠癌,由IBD发展而来的肠癌称为肠炎相关结直肠癌(CAC)。IBD患者肠上皮屏障破损,肠道微生物进入肠粘膜固有层,激活固有免疫细胞Toll样受体(TLRs)炎性通路,如巨噬细胞TLR4炎性通路,释放大量促炎因子和DNA突变诱导剂活性氧(ROS),诱导肠上皮细胞恶性变,从而导致肠癌发生发展。肠道炎性微环境是CAC发生的重要诱因,抑制肠道炎症是防治CAC的重要手段。氯喹(CQ)及其衍生物羟氯喹(HCQ)除治疗疟疾外,也用于治疗炎症性疾病,如细菌感染、系统性红斑狼疮、风湿性关节炎等。CQ/HCQ通过抑制胞内细菌生长、促炎因子释放、促炎信号通路活化发挥抗炎作用。此外,CQ/HCQ也能用于治疗肿瘤,现有多项临床试验表明:CQ/HCQ通过抑制肿瘤细胞增殖、促进凋亡,治疗非小细胞肺癌、头颈部肿瘤和转移性实体瘤等。有研究发现CQ能有效缓解轻中度活动性UC患者肠道炎症,但是,CQ/HCQ对IBD诱导而来CAC是否有防治作用未知,值得我们进一步探讨。目的:在小鼠致死性肠炎和CAC模型中,研究HCQ对肠炎和肠道肿瘤的疗效,并对其抗炎和抗肿瘤机制进行初步探讨。方法:1.用高剂量葡聚糖硫酸钠(DSS)喂予小鼠建立致死性肠炎模型,HCQ持续每天给药,观察小鼠存活率。2.用氧化偶氮甲烷(AOM)+DSS建立小鼠早期CAC模型,HCQ持续每天给药,评估小鼠肠炎。分离纯化小鼠肠粘膜固有层免疫细胞,Westernblot检测炎性蛋白NF-κB、STAT3和MAPK表达,实时定量PCR检测促炎因子IL1β、IL6、TNFα、COX2、IFNβ、IP10、RANTES 和 MCP1 的 mRNA 水平,流式检测ROS表达。3.分析基因表达综合数据库(GEO)微阵列数据,研究TLR4基因在IBD患者和健康人群表达变化。4.体外TLR4配体脂多糖刺激巨噬细胞活化,检测HCQ对TLR4信号通路蛋白表达、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持续每天给药,观察小鼠肠道肿瘤数目、直径改变。分离纯化小鼠肠粘膜固有层免疫细胞,Western blot检测炎性蛋白表达。分离纯化小鼠肠道肿瘤细胞,免疫组化检测细胞增殖蛋白Ki-67、凋亡蛋白c-PARP表达,Western blot检测细胞周期调控蛋白、凋亡蛋白表达,免疫荧光染色分析小鼠结肠Tunel表达。结果:1.HCQ显著提高小鼠致死性肠炎存活率。2.HCQ缓解小鼠CAC早期肠道炎症,具体表现为缓解小鼠体重下降、结肠缩短,降低疾病活动指数和组织形态学评分。HCQ抑制肠固有层免疫细胞炎性蛋白 NK-κB、STAT3 和 MAPK 磷酸化、促炎因子 IL1 β、IL6、TNFα、COX2、IFNβ、IP10、RANTES 和 MCP1 表达、ROS 释放。3.与健康人群相比,IBD患者肠道高表达TLR4基因。体外实验,HCQ抑制巨噬细胞TLR4信号通路,具体表现为抑制TLR4通路相关蛋白活化,抑制促炎因子表达和ROS释放。4.HCQ抑制小鼠CAC中晚期肠道肿瘤发生发展,具体表现为缓解小鼠体重下降,小鼠肠道肿瘤数目减少、直径减小,降低小鼠腺癌比例。HCQ抑制肠固有层免疫细胞炎性蛋白NK-κB、STAT3和MAPK磷酸化。HCQ抑制肠癌细胞增殖、诱导周期停滞和细胞凋亡。结论:本课题中,我们发现HCQ缓解小鼠CAC模型早期肠炎和中晚期肿瘤发生发展。在小鼠CAC早期肠炎,HCQ抑制肠固有层免疫细胞炎性通路活化、促炎因子生成和DNA突变诱导剂ROS释放。HCQ发挥抗炎作用可能部分是通过抑制巨噬细胞TLR4通路活化起作用的。因此,HCQ可以抑制肠炎,进而抑制肠上皮细胞恶性变,抑制肿瘤发生发展。此外,HCQ也能直接调控肿瘤细胞。在小鼠中晚期CAC,HCQ能抑制肠道肿瘤细胞增殖、促进其周期停滞和凋亡。总之,HCQ通过抗炎和抗肿瘤双重作用,对小鼠CAC起防治作用。临床上,HCQ可作为潜在药物,防止IBD向CAC发生发展。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.34;R574

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