CHL1在炎症性肠病中的作用及其机制
发布时间：2019-07-02 09:30
【摘要】：炎症性肠病(Inflammatory bowel disease,IBD)包括溃疡性结肠炎(ulcerative colitis)和克罗恩病(Crohn’s disease),这是一种胃肠道的炎症性疾病,伴随着严重的病理学变化,预后不佳且病因未明。溃疡性结肠炎的炎症主要发生在粘膜当中。最主要的症状包括腹泻,结肠直肠出血,粘液增多和腹痛。克罗恩病是一种慢性炎症,从口到肛门的消化道都可能发生,主要发生在回肠,盲肠和结肠。在克罗恩病的发生中,炎症从肠道的粘膜层穿透到浆膜层,具有节段性不对称的特征,在某些病人身上可能发现上皮样肉芽肿。IBD在欧美国家高发,但根据最新的研究,在亚洲和南美洲,IBD的患病率也日益增高。IBD的病因主要与环境、免疫、遗传等因素有关,但是,慢性炎症在肠道反复发作的机制尚不清楚。部分观点认为,肠道慢性炎症的发生与肠道粘膜免疫缺陷有关[28]。近年来的研究发现,提示细胞黏附分子在肠道炎症发展的过程中起重要作用。几种主要的细胞黏附分子介导了淋巴细胞的募集,包括选择素,整合素,钙黏蛋白和免疫球蛋白超家族。L-选择素或P-选择素使得白细胞被高内皮小静脉捕获贴壁,通过滚动,LFA-1与整合素之间形成牢固黏附,α4β1(VLA-4)整合素、α4β7整合素与上皮细胞上的VCAM-1和MAdCAM-1形成牢固黏附。临床上诸多药物是通过抑制细胞黏附分子(cell adhesion molecule,CAM)对炎症细胞的结合实现的。比如抑制α4β1 VCAM之间形成牢固黏附的那他珠单抗,抑制α4β7-MAdCAM-1形成牢固黏附的委地落珠单抗。CHL1是一种在人体内普遍表达的神经细胞黏附分子(Neural cell adhesion molecule,NCAM),在以往的研究中,发现它对成人神经回路轴突形成、神经元的生长和存活有着至关重要的作用。另一方面,CHL1与结直肠癌删除分子(Deleted in colorectal carcinoma,DCC)有相似的结构域。有研究表明,CHL1在结肠癌病人的结肠组织中表达降低[8,9]。但CHL1与炎症性肠病之间的关系尚缺乏探讨。炎症性肠病的发生往往伴随着体内炎症因子的增加,以及炎症细胞对肠粘膜上皮的浸润。中性粒细胞的浸润在溃疡性结肠炎的发生中,起着关键性的作用,也是衡量疾病活动度以及炎症程度的关键标志。正常健康组织中,中性粒细胞在粘膜固有层少量分布,随着炎症进行,浸润到上皮表面或结肠隐窝,与隐窝的退行性变化和隐窝浸润有关。IBD的活动度与中性粒细胞浸润的发展、隐窝消失呈正相关。所以在对于IBD的干扰治疗中,中性粒细胞被认为是一种可能的药理学靶标。在IBD中,由于炎症反应的增强,单核细胞来源的巨噬细胞往往会增多,巨噬细胞对维持胃肠道环境内稳态起至关重要,同时可以抵制病原体破坏,以保护肠道。巨噬细胞浸润程度也是一种反应炎症程度的标志。本研究利用葡聚糖硫酸钠(Dextran Sulfate Sodium,DSS)诱导的小鼠结肠炎模型,探讨了CHL1缺失对DSS诱导的小鼠结肠炎症的影响,研究中性粒细胞和巨噬细胞浸润对促进结肠炎症反应和组织损伤的机制。主要的研究结果如下:1.炎症性肠病上调小鼠结肠组织CHL1的表达为了探索炎症性肠病与神经黏附分子CHL1的关系,建立经典的DSS小鼠炎症性肠病模型。在DSS诱导炎症性肠病的第5天,第7天,第9天,检测小鼠结肠组织CHL1组织的表达。在蛋白水平上,从诱导炎症性肠病的第5天开始,CHL1的表达上升,第7天和第9天CHL1的表达持续性增高。在转录水平,实时定量PCR(Q-PCR)检测CHL1 mRNA的表达。从第7天开始,CHL1在mRNA水平的表达显著增加。这说明不论在转录水平还是蛋白表达水平,炎症性肠病小鼠结肠组织CHL1的表达都是上调的。2.CHL1缺失加重炎症性肠病的发生为了进一步探索CHL1与炎症性肠病的关系,将CHL1(+/+)型和CHL1(-/-)型小鼠根据是否喂养DSS分为四组,建立炎症性肠病模型。发现与DSS诱导的CHL1(+/+)型小鼠相比,DSS诱导的CHL1(-/-)型小鼠在炎症性肠病的发生中便血明显,从应激的第7天开始,体重明显减轻,在第9天,DSS诱导的CHL1(-/-)型小鼠死亡率明显增加。检测小鼠肠组织,DSS诱导的CHL1(-/-)型小鼠结肠长度明显缩短,炎细胞浸润和组织损伤严重。以上结果反映了CHL1缺失加重了炎症性肠病的发生。3.CHL1缺失引起小鼠结肠上皮细胞结构的改变,增加肠道的通透性观察小鼠结肠上皮组织形态学的变化,发现CHL1缺失可以使原本排列紧密的小鼠结肠柱状上皮细胞排列变得较为松散,细胞体积明显增大,杯状细胞增多。在DSS应激后,DSS诱导的CHL1(+/+)型小鼠的上皮细胞排列仍旧有序,而DSS诱导的CHL1(-/-)型小鼠的柱状上皮细胞排列明显紊乱。CHL1(-/-)型小鼠结肠上段的改变以上皮细胞细胞形态的改变为主。结肠下段的改变以上皮细胞细胞形态的紊乱为主。结肠中段的改变介于上段和下段的改变之间。通过FITC检测肠道通透性,喂养DSS的CHL1(-/-)型小鼠肠道通透性明显升高。结肠末端对FITC的截留都是最强的,DSS诱导的CHL1(-/-)型小鼠对FITC的截留与DSS诱导的CHL1(+/+)型小鼠相比无明显改变,而DSS诱导的CHL1(-/-)型小鼠结肠上端、中段对FITC的截留要明显强于DSS诱导的CHL1(-/-)型小鼠。4.CHL1缺失增加结肠组织中炎症细胞的浸润为了检测CHL1缺失影响炎症性肠病的免疫学机制,DSS诱导第5天,第7天检测外周血中性粒细胞的表达,发现与喂养DSS的CHL1(+/+)型小鼠相比,DSS诱导的CHL1(-/-)型小鼠模型外周血中性粒细胞的数量并不增高,表明CHL1缺失并不会促进小鼠外周血中中心粒细胞的增殖分化,但是小鼠结肠组织中中性粒细胞的浸润明显增多,这种变化很可能是因为CHL1的缺失导致。此外,在结肠组织内,巨噬细胞对结肠组织浸润也明显增多。综上所述,本研究首次观察到神经粘附分子CHL1在肠道组织中表达,并且在炎症性肠病时CHL1的表达明显升高;CHL1缺失后通过增强炎症细胞的浸润和肠道的通透性,加重炎症性肠病的发生。以上结果提示CHL1在炎症性肠病的发生中发挥重要保护作用。该研究为炎症性肠病的干预提供了新的思路。
[Abstract]:Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease, is an inflammatory disease in the gastrointestinal tract, with severe pathological changes, poor prognosis and an unknown cause. The inflammation of the ulcerative colitis mainly occurs in the mucous membrane. The most important symptoms include diarrhea, colorectal bleeding, increased mucus, and abdominal pain. Crohn's disease is a chronic inflammation that can occur in the digestive tract from the mouth to the anus, mainly in the ileum, the cecum and the colon. In the occurrence of Crohn's disease, inflammation is penetrated from the mucosal layer of the intestinal tract to the serosa layer, with the characteristics of segmental asymmetry, and epithelial-like granuloma may be found on some patients. IBD is a high incidence in the European and American countries, but the prevalence of IBD is also increasing in Asia and South America, according to the latest study. The etiology of IBD is mainly related to the factors such as environment, immunity, and genetics. However, the mechanism of chronic inflammation in the intestine is not clear. In some view, the occurrence of chronic inflammation of the intestinal tract is related to the intestinal mucosal immune deficiency[28]. In recent years, it has been found that cell adhesion molecules play an important role in the development of intestinal inflammation. Several major cell adhesion molecules mediate the recruitment of lymphocytes, including the selection of the protein, the integrin, the calcium mucin and the immunoglobulin superfamily. L-selectin or P-selectin was used to capture the adherence of the white blood cells to the high-endothelial venules, and a strong adhesion was formed between the LFA-1 and the integrin by rolling, and the VCAM-1 and the MAdCAM-1 on the epithelial cells were firmly adhered to the total of 4-1 (VLA-4) integrin. In clinical, many drugs are achieved by inhibiting the binding of cell adhesion molecules (CAM) to the inflammatory cells. For example, to inhibit the formation of a strong adhesion of the bevacizumab between the babo4 and 1vcam, and to inhibit the formation of a firm-adhered monocytic bevacizumab in the h4-7-mAdCAM-1. CHL1 is a kind of nerve cell adhesion molecule (NCAM) which is widely expressed in human body. In the past study, it has been found that it plays an important role in the formation of axons of adult neural circuits, the growth and survival of neurons. On the other hand, CHL1 and the deletion of colorectal cancer (DCC) have similar domains. Studies have shown that the expression of CHL1 in the colon tissue of patients with colon cancer is reduced[8,9]. However, the relationship between CHL1 and inflammatory bowel disease is still lacking. The occurrence of inflammatory bowel disease is often accompanied by an increase in inflammatory factors in the body, as well as the infiltration of inflammatory cells on the intestinal mucosa epithelium. The infiltration of neutrophils plays a key role in the occurrence of ulcerative colitis, and is a key marker to measure the activity of the disease and the degree of inflammation. In the normal healthy tissue, the neutrophils are distributed in a small amount in the lamina propria, and with the inflammation, infiltration to the epithelial surface or the crypt of the colon is related to the degenerative changes of the crypt and the infiltrates of the crypt. The activity of IBD is related to the development of neutrophil infiltration and the disappearance of the crypt. Neutrophil is considered a potential pharmacological target in the treatment of the interference with IBD. In IBD, monocyte-derived macrophages tend to increase due to the increased inflammatory response, and macrophages are critical to maintaining a steady state in the gastrointestinal environment, while resisting the destruction of pathogens to protect the intestinal tract. The degree of macrophage infiltration is also a sign of the degree of inflammation. In this study, the effect of CHL1 deletion on the colon inflammation induced by DSS was discussed by using Dextran Sulfonate Sodidium (DSS), and the mechanism of the infiltration of neutrophils and macrophages on the inflammatory response and tissue injury of the colon was studied. The main results are as follows:1. In order to explore the relationship between the inflammatory bowel disease and the neuroadhesion molecule CHL1, the expression of CHL1 in the colon tissue of the mouse was up-regulated by inflammatory bowel disease, and the model of the classic DSS mouse inflammatory bowel disease was established. The expression of the CHL1 tissue in the colon tissue of the mouse was detected on Day 5, Day 7, and Day 9 of the DSS-induced inflammatory bowel disease. At the protein level, the expression of CHL1 increased from day 5 of the induction of inflammatory bowel disease, and the expression of CHL1 on Day 7 and Day 9 continued to increase. The expression of CHL1 mRNA was detected by real-time quantitative PCR (Q-PCR) at the level of transcription. From day 7 onwards, the expression of CHL1 at the mRNA level was significantly increased. in ord to further explore that relationship between CHL1 and inflammatory bowel disease, The CHL1 (+/ +) and CHL1 (-/-) mice were divided into four groups according to whether or not the DSS was fed, and an inflammatory bowel disease model was established. In comparison with DSS-induced CHL1 (+/ +)-type mice, DSS-induced CHL1 (-/-)-type mice were significantly reduced in the occurrence of inflammatory bowel disease, with a significant reduction in body weight from day 7 of stress, and a significant increase in the mortality of the CHL1 (-/-)-type mice induced by DSS on Day 9. The length of the colon of the CHL1 (-/-) type mouse induced by DSS was significantly shortened, and the infiltration of the inflammatory cells and the tissue damage were serious. The results indicated that the deletion of CHL1 aggravated the occurrence of inflammatory bowel disease.3. The deletion of CHL1 caused the change of the structure of the colon epithelial cells in the mice, and the changes of the morphology of the epithelial tissue of the colon of the mice were observed by increasing the permeability of the intestinal tract. It was found that the deletion of CHL1 could make the arrangement of the columnar epithelial cells of the colon of the mouse in close alignment to be loose, the volume of the cells increased obviously, and the goblet-like cells increased. After DSS stress, the arrangement of the epithelial cells of the DSS-induced CHL1 (+/ +)-type mice is still in order, while the columnar epithelial cells of the DSS-induced CHL1 (-/-)-type mice are in an obvious disorder. The changes of the upper segment of the colon of the CHL1 (-/-) type mouse were mainly the change of the cell morphology of the epithelial cells. The changes of the lower segment of the colon were dominated by the disorder of the cell morphology of the epithelial cells. The change in the middle of the colon is between the changes in the upper and lower segments. The intestinal permeability of the CHL1 (-/-) type mice fed with DSS was significantly increased by the detection of intestinal permeability with FITC. The concentration of FITC at the end of the colon was the strongest, and the retention of the DSS-induced CHL1 (-/-)-type mouse did not change significantly as compared to the DSS-induced CHL1 (+/ +)-type mouse, and the DSS-induced upper end of the CHL1 (-/-)-type mouse colon, 4. ChL1 deletion increased the infiltration of inflammatory cells in the colon tissue in order to detect the immunological mechanism of CHL1 deletion on inflammatory bowel disease, DSS induced the fifth day and day 7 to detect the expression of peripheral blood neutrophils. Compared with the CHL1 (+/ +)-type mice fed with DSS, the number of peripheral neutrophils in the DSS-induced CHL1 (-/-)-type mouse model was not increased, indicating that the deletion of CHL1 did not promote the proliferation and differentiation of the central granulocytes in the peripheral blood of the mouse, However, the infiltration of neutrophils in the colon tissue of the mice is significantly increased, which is likely due to the absence of CHL1. In addition, in that colon tissue, the infiltration of the macrophages to the colon is also significantly increase. In conclusion, the first time in this study, the expression of CHL1 in the intestinal tissue was observed, and the expression of CHL1 in inflammatory bowel disease was significantly increased; after CHL1 deletion, the infiltration of inflammatory cells and the permeability of the intestinal tract were enhanced, and the occurrence of inflammatory bowel disease was increased. The above results suggest that CHL1 plays an important protective role in the occurrence of inflammatory bowel disease. The study provided a new way of thinking for the intervention of inflammatory bowel disease.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R574
本文编号：2508808
[Abstract]:Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease, is an inflammatory disease in the gastrointestinal tract, with severe pathological changes, poor prognosis and an unknown cause. The inflammation of the ulcerative colitis mainly occurs in the mucous membrane. The most important symptoms include diarrhea, colorectal bleeding, increased mucus, and abdominal pain. Crohn's disease is a chronic inflammation that can occur in the digestive tract from the mouth to the anus, mainly in the ileum, the cecum and the colon. In the occurrence of Crohn's disease, inflammation is penetrated from the mucosal layer of the intestinal tract to the serosa layer, with the characteristics of segmental asymmetry, and epithelial-like granuloma may be found on some patients. IBD is a high incidence in the European and American countries, but the prevalence of IBD is also increasing in Asia and South America, according to the latest study. The etiology of IBD is mainly related to the factors such as environment, immunity, and genetics. However, the mechanism of chronic inflammation in the intestine is not clear. In some view, the occurrence of chronic inflammation of the intestinal tract is related to the intestinal mucosal immune deficiency[28]. In recent years, it has been found that cell adhesion molecules play an important role in the development of intestinal inflammation. Several major cell adhesion molecules mediate the recruitment of lymphocytes, including the selection of the protein, the integrin, the calcium mucin and the immunoglobulin superfamily. L-selectin or P-selectin was used to capture the adherence of the white blood cells to the high-endothelial venules, and a strong adhesion was formed between the LFA-1 and the integrin by rolling, and the VCAM-1 and the MAdCAM-1 on the epithelial cells were firmly adhered to the total of 4-1 (VLA-4) integrin. In clinical, many drugs are achieved by inhibiting the binding of cell adhesion molecules (CAM) to the inflammatory cells. For example, to inhibit the formation of a strong adhesion of the bevacizumab between the babo4 and 1vcam, and to inhibit the formation of a firm-adhered monocytic bevacizumab in the h4-7-mAdCAM-1. CHL1 is a kind of nerve cell adhesion molecule (NCAM) which is widely expressed in human body. In the past study, it has been found that it plays an important role in the formation of axons of adult neural circuits, the growth and survival of neurons. On the other hand, CHL1 and the deletion of colorectal cancer (DCC) have similar domains. Studies have shown that the expression of CHL1 in the colon tissue of patients with colon cancer is reduced[8,9]. However, the relationship between CHL1 and inflammatory bowel disease is still lacking. The occurrence of inflammatory bowel disease is often accompanied by an increase in inflammatory factors in the body, as well as the infiltration of inflammatory cells on the intestinal mucosa epithelium. The infiltration of neutrophils plays a key role in the occurrence of ulcerative colitis, and is a key marker to measure the activity of the disease and the degree of inflammation. In the normal healthy tissue, the neutrophils are distributed in a small amount in the lamina propria, and with the inflammation, infiltration to the epithelial surface or the crypt of the colon is related to the degenerative changes of the crypt and the infiltrates of the crypt. The activity of IBD is related to the development of neutrophil infiltration and the disappearance of the crypt. Neutrophil is considered a potential pharmacological target in the treatment of the interference with IBD. In IBD, monocyte-derived macrophages tend to increase due to the increased inflammatory response, and macrophages are critical to maintaining a steady state in the gastrointestinal environment, while resisting the destruction of pathogens to protect the intestinal tract. The degree of macrophage infiltration is also a sign of the degree of inflammation. In this study, the effect of CHL1 deletion on the colon inflammation induced by DSS was discussed by using Dextran Sulfonate Sodidium (DSS), and the mechanism of the infiltration of neutrophils and macrophages on the inflammatory response and tissue injury of the colon was studied. The main results are as follows:1. In order to explore the relationship between the inflammatory bowel disease and the neuroadhesion molecule CHL1, the expression of CHL1 in the colon tissue of the mouse was up-regulated by inflammatory bowel disease, and the model of the classic DSS mouse inflammatory bowel disease was established. The expression of the CHL1 tissue in the colon tissue of the mouse was detected on Day 5, Day 7, and Day 9 of the DSS-induced inflammatory bowel disease. At the protein level, the expression of CHL1 increased from day 5 of the induction of inflammatory bowel disease, and the expression of CHL1 on Day 7 and Day 9 continued to increase. The expression of CHL1 mRNA was detected by real-time quantitative PCR (Q-PCR) at the level of transcription. From day 7 onwards, the expression of CHL1 at the mRNA level was significantly increased. in ord to further explore that relationship between CHL1 and inflammatory bowel disease, The CHL1 (+/ +) and CHL1 (-/-) mice were divided into four groups according to whether or not the DSS was fed, and an inflammatory bowel disease model was established. In comparison with DSS-induced CHL1 (+/ +)-type mice, DSS-induced CHL1 (-/-)-type mice were significantly reduced in the occurrence of inflammatory bowel disease, with a significant reduction in body weight from day 7 of stress, and a significant increase in the mortality of the CHL1 (-/-)-type mice induced by DSS on Day 9. The length of the colon of the CHL1 (-/-) type mouse induced by DSS was significantly shortened, and the infiltration of the inflammatory cells and the tissue damage were serious. The results indicated that the deletion of CHL1 aggravated the occurrence of inflammatory bowel disease.3. The deletion of CHL1 caused the change of the structure of the colon epithelial cells in the mice, and the changes of the morphology of the epithelial tissue of the colon of the mice were observed by increasing the permeability of the intestinal tract. It was found that the deletion of CHL1 could make the arrangement of the columnar epithelial cells of the colon of the mouse in close alignment to be loose, the volume of the cells increased obviously, and the goblet-like cells increased. After DSS stress, the arrangement of the epithelial cells of the DSS-induced CHL1 (+/ +)-type mice is still in order, while the columnar epithelial cells of the DSS-induced CHL1 (-/-)-type mice are in an obvious disorder. The changes of the upper segment of the colon of the CHL1 (-/-) type mouse were mainly the change of the cell morphology of the epithelial cells. The changes of the lower segment of the colon were dominated by the disorder of the cell morphology of the epithelial cells. The change in the middle of the colon is between the changes in the upper and lower segments. The intestinal permeability of the CHL1 (-/-) type mice fed with DSS was significantly increased by the detection of intestinal permeability with FITC. The concentration of FITC at the end of the colon was the strongest, and the retention of the DSS-induced CHL1 (-/-)-type mouse did not change significantly as compared to the DSS-induced CHL1 (+/ +)-type mouse, and the DSS-induced upper end of the CHL1 (-/-)-type mouse colon, 4. ChL1 deletion increased the infiltration of inflammatory cells in the colon tissue in order to detect the immunological mechanism of CHL1 deletion on inflammatory bowel disease, DSS induced the fifth day and day 7 to detect the expression of peripheral blood neutrophils. Compared with the CHL1 (+/ +)-type mice fed with DSS, the number of peripheral neutrophils in the DSS-induced CHL1 (-/-)-type mouse model was not increased, indicating that the deletion of CHL1 did not promote the proliferation and differentiation of the central granulocytes in the peripheral blood of the mouse, However, the infiltration of neutrophils in the colon tissue of the mice is significantly increased, which is likely due to the absence of CHL1. In addition, in that colon tissue, the infiltration of the macrophages to the colon is also significantly increase. In conclusion, the first time in this study, the expression of CHL1 in the intestinal tissue was observed, and the expression of CHL1 in inflammatory bowel disease was significantly increased; after CHL1 deletion, the infiltration of inflammatory cells and the permeability of the intestinal tract were enhanced, and the occurrence of inflammatory bowel disease was increased. The above results suggest that CHL1 plays an important protective role in the occurrence of inflammatory bowel disease. The study provided a new way of thinking for the intervention of inflammatory bowel disease.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R574
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相关期刊论文 前4条
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