【作者】 王丹；

【导师】 邱春红；

【作者基本信息】 山东大学 ， 细胞生物学， 2016， 硕士

【摘要】 研究背景：炎症性肠疾病(Inflammatory bowel disease, IBD)包括克罗恩病(Crohn’s disease, CD)和溃疡性结肠炎(Ulcerative colitis, UC). IBD的发病率逐年升高,而且,IBD患者形成结直肠癌(CRC)的风险会增加,因此受到了人们的广泛关注。目前,IBD的发病机制仍不清楚,但是遗传、免疫和环境风险等因素的相互作用与IBD的发病机制具有一定关联。巨噬细胞作为一种重要的免疫细胞在炎症疾病中具有非常重要的作用。巨噬细胞是高度异质性的细胞,许多组织及腔室都分布着多种具有不同表型及功能的巨噬细胞。CD169(Siglec-1,唾液酸粘附素)是表达在特定巨噬细胞表面的唾液酸粘附受体。在淋巴结中,CD169+巨噬细胞能够通过吞噬死亡肿瘤细胞并随后将肿瘤相关抗原提呈至CD8+T细胞而介导抗肿瘤免疫。在脾脏中,CD169+巨噬细胞位于脾脏白髓的边缘区,可以通过吞噬凋亡细胞并诱导免疫耐受调控机体的免疫平衡。在肠道及腹腔都分布着一类表达CD169的巨噬细胞。有研究报道,肠固有层定居的CD169+巨噬细胞与黏膜损伤及炎性单核细胞招募有关。腹腔巨噬细胞在控制炎症和防御腹腔感染等方面也具有非常重要的作用。由于腹腔巨噬细胞与肠道定居巨噬细胞的起源不同,因此,研究腹腔中表达CD169的巨噬细胞在肠黏膜炎症中的作用有助于理解IBD的发病机制,为IBD炎症的控制和缓解提供新的思路。研究目的：通过使用葡聚糖硫酸钠(DSS)建立的小鼠结肠炎模型和CD169-DTR (diphtheria toxin receptor,白喉毒素受体)转基因小鼠来研究小鼠腹腔CD169+巨噬细胞在结肠炎症发展中的作用,并进一步研究CD169+巨噬细胞在DSS诱导结肠炎中的具体作用机制。研究方法：首先用流式和免疫荧光染色的方法确定了CD11b+CD169+腹腔巨噬细胞在DSS诱导结肠炎症中的数量变化,提示了CD169+腹腔巨噬细胞在结肠炎中发挥重要作用。然后,使用CD169-DTR转基因小鼠进一步研究CD169+巨噬细胞在DSS诱导结肠炎中的作用。将白喉毒素(DT)注射到CD169-DTR、鼠体内后,CD169+巨噬细胞选择性缺失。利用DSS诱导的结肠炎模型,分别比较C57BL/6野生型小鼠和CD169-DTR、鼠的临床发病情况。通过比较两种小鼠的体重变化、结肠长度及结肠组织结构形态的变化判断CD169+腹腔巨噬细胞缺失后小鼠的发病情况,明确该细胞亚群的存在与否对于结肠炎发病的重要性。此外还通过Q-PCR和ELISA等分子生物学方法检测了CD169+巨噬细胞缺失后,相关炎性因子和趋化因子的表达变化,进一步明确了CD169+腹腔巨噬细胞在结肠炎中具体的作用机制。实验结果：在DSS诱导的结肠炎中,CD11b+CD169+腹腔巨噬细胞的数量明显增加,提示CD169+腹腔巨噬细胞在结肠炎的炎症过程中有重要作用。CD169-DTR小鼠对比实验表明CD169+巨噬细胞缺失后的小鼠不能被DSS诱导典型的结肠炎。与野生型小鼠相比,CD169-DTR、鼠的体重减轻和直肠出血等结肠炎症状并不明显。而且,组织学结果显示结肠组织的炎性细胞浸润及损伤情况程度也明显不如野生型小鼠。Q-PCR结果显示IL-1β、IL-6、IL-12、IL-23和TNFα等炎性因子的表达水平明显低于野生型小鼠。此外,趋化因子的分析结果显示DSS诱导的野生型小鼠肠炎模型中,腹腔巨噬细胞表达CCL3、CCL8的表达水平降低,而CCL22的表达明显升高。对野生型小鼠连续6天注射重组CCL22蛋白后,DSS诱导结肠炎的症状得到明显缓解。结论：本实验揭示了腹腔CD169+巨噬细胞在DSS诱导结肠炎中起着重要作用。腹腔CD169+巨噬细胞的数量在CD169-DTR小鼠注射DT后,CD169+巨噬细胞选择性缺失,使DSS诱导结肠炎过程中CCL22的表达升高。向DSS诱导的小鼠体内注射CCL22重组蛋白治疗结肠炎,能使结肠炎的临床症状得到缓解。CD169+巨噬细胞可能是通过调控CCL22的分泌而在DSS诱导结肠炎中发挥作用,表明CCL22有可能成为抗炎治疗的佐剂。更多还原

【Abstract】 Background:Crohn’s disease (CD) and ulcerative colitis (UC) are the most widely known types of IBD and have been the focus of attention due to their increasing incidence. The correlation between inflammation and the development of cancer has been confirmed by many studies, and IBD is also related to a substantial increase in the risk of colorectal cancer (CRC). At present, although the factors that contribute to the development of IBD and the pathogenesis have not been fully elucidated, an interaction between genetic, immune and environmental risk factors is thought to play a role in the pathogenesis of IBD.Macrophages, known for their role as immune sentinels, play a critical role in inflammation disease. Macrophages are highly heterogeneous cells that many tissues and serous cavities contain multiple types of macrophages with distinct phenotypes and functions. CD 169 (also known as sialoadhesin, Siglec-1) is a cell surface sialic-acid-binding receptor, which is expressed by specific macrophage subsets. In lymph node, CD169+ macrophages dominate antitumor immunity by phagocytosing dead tumor cells transported via lymphatic flow and crosspresenting tumor antigens to CD8+ T cells subsequently. In spleen, CD169+ macrophages that located in the marginal zone (MZ) have a role in inducing immune tolerance. It has been reported a LP-resident macrophage subset expressing CD169 links mucosal damage and inflammatory monocyte recruitment. Peritoneal macrophages also play important roles in the control of inflammatory pathologies, as well as in the defense against infection of the abdominal cavity. However, whether a peritoneal macrophage expressing CD 169 plays important role in mucosal inflammation has not been reported yet.Research purpose:To study the role of a peritoneal CD169-expressing macrophages subset during the development of inflammation by using Dextran sulphate sodium (DSS) induced colitis and CD169-DTR (diphtheria toxin receptor) transgenic mice, and try to investigate the cellular and molecular mechanism of IBD mediated by CD 169 expressing cells in peritoneal cavity.Methods:Firstly, the role of CD11b+CD169+ macrophages from peritoneal cavity was examined by FACS and immunofluorescence histochemistry during the inflammatory of DSS-induced colitis. Next, the role of peritonealCD169+ macrophages in DSS induced colitis was studied by using the CD169-human diphtheria toxin receptor (DTR) transgenic mice in which CD169+ macrophages could be deleted by diphtheria toxin (DT) injection. Wild type (WT) mice and CD169-DTR mice were administrated orally with 3.5% DSS in drinking water for 7 days to induce colitis. The severity of colitis was assessed by body weight, colon length and histological examination. And the expression levels of inflammatory cytokines and chemokines in the peritoneal macrophages were also detected by Q-PCR and ELISA both in WT mice and DT-injected CD169-DTR mice. At last, the potential effect of CCL22 for therapy of IBD was explored by using DSS-induced colitis.Results:Both the results of FACS and histological analysis showed that the numbers of CD11b+CD169+ macrophages increased significantly during the DSS-induced colitis and these results strongly indicated that peritoneal macrophages expressing CD 169 were exactly involved in the UC. Moreover, DSS-induced colitis was alleviated in CD169-DTR mice at least partially due to the deletion CD169+ macrophages. The symptoms of colitis such as diarrhea, rectal bleeding and weight loss were not significant in CD169-DTR mice than those in WT mice and the expression levels of inflammatory cytokines such as IL-1β、IL-6、IL-12、IL-23 and TNFa decreased significantly in CD169-DTR mice than those in WT mice. Histological examination of the colons from CD169-DTR mice showed a lower degree of mucosal injury and greatly reduced numbers of infiltrating inflammatory cells. Furthermore, the CCL22 expression level in peritoneal macrophages from CD169-DTR mice was much higher than that from WT mice in DSS-induced colitis. And, a total of six daily subcutaneous injections of rmCCL22 during the DSS-induced colitis ameliorated clinical symptoms of colitis.Conclusions:The present study demonstrated peritoneal CD169+ macrophages play critical role in DSS-induced colitis. CD169+ macrophages transiently deleted by DT injection caused higher expression of CCL22 in DSS-induced colitis. The in vivo treatment with rmCCL22 ameliorated the clinical symptoms of DSS-induced colitis. CD169+ macrophages may influence the DSS-induced colitis by dominantly the secreting of CCL22. CCL22 could be potentially used as a preventive agent or as an adjuvant in anti-inflammatory therapy.更多还原