TL1A对慢性实验性结肠炎小鼠结肠组织Th9细胞活化的影响
发布时间：2018-07-26 16:23
【摘要】：溃疡性结肠炎(ulcerative colitis, UC)是炎症性肠病(inflammatorybowel disease, IBD)的一种，其发病机制尚不明了。目前认为肠黏膜免疫异常、持续肠道感染、遗传和环境等因素均参与了该病的发生发展，其中免疫调控失衡是UC的重要致病因素。CD4+T细胞是免疫系统中最重要的效能细胞，包括辅助性T细胞(T helper cell, Th cell)和调节性T细胞(Tregulatory T cell, Treg)，在调控体内免疫应答和维持免疫平衡中发挥重要作用。CD4+T细胞过度增殖、分化导致促炎与抗炎免疫失衡是UC的主要特点之一。 近年来的研究表明，转化生长因子β(transforming growth factor-β,TGF-β)和白细胞介素(interleukin, IL)-4联合可诱导CD4+初始T细胞分化成一群大量分泌IL-9的新T细胞亚群，活化的Th2细胞在TGF-β单独存在的情况下也可以产生相同的结果。因其主要分泌细胞因子IL-9，且诱导分化途径又不同于T细胞的其他亚群，所以被命名为Th9(CD4+IL-9+)细胞。Th9细胞参与了自身免疫性脑脊髓膜炎、结核性胸膜炎、过敏性皮炎等多种炎性疾病的炎症进展，给重组活化基因1缺陷(recombination-activating gene1-deficient, RAG-1)小鼠回输Th9细胞亦可成功诱导小鼠结肠炎症。 肿瘤坏死因子样配体1A(tumor necrosis factor-like ligand1aberrance,TL1A)是肿瘤坏死因子超家族成员15(TNF superfamily member15,TNFSF15)基因的蛋白产物，主要表达在人类的内皮细胞，包括人脐静脉内皮细胞、成人皮肤微血管内皮细胞和子宫肌层内皮细胞等。目前研究表明，TL1A是IBD的易感基因，可通过与死亡受体3(death receptor, DR3)结合，激活Th1、Th2和Th17细胞，致肠黏膜免疫紊乱，引发IBD的肠道炎症反应。然而目前TL1A对Th9细胞的影响尚不清楚。本研究主要是通过检测TL1A转基因小鼠及WT小鼠结肠组织中Th9细胞水平探讨TL1A对Th9细胞活化的影响。 目的：探讨TL1A对慢性实验性结肠炎小鼠结肠组织中Th9细胞活化的影响。 方法：①本实验以淋巴细胞过表达TL1A的转基因小鼠及WT小鼠为研究对象，采用RT-PCR方法进行TL1A的转基因(LCK-CD2-TL1A-GFP,L-Tg)小鼠的鉴定与筛选；②12只具有相同遗传背景的C57BL/6WT小鼠(体重15~18g，6~8周龄)随机分为Control/WT组、DSS/WT组，每组6只；12只L-Tg小鼠(体重15~18g，6~8周龄)随机分为Control/Tg组、DSS/Tg组，每组6只。Control组饮用蒸馏水，DSS组间断饮用3%右旋葡聚糖硫酸钠(dextran sulfate sodium, DSS)，时间段分别为第1~5天、第8~12天、第15~19天、第22~26天及第27、28天，其余时间饮用蒸馏水，第29天处死动物；③每日观察小鼠的精神状态、体重、活动情况、毛发光泽度、粪便形状、大便潜血情况等，进行疾病活动指数(disease activityindex, DAI)评分，评估结肠炎症程度；④观察结肠形态学改变，记录结肠长度变化，，进行大体评分；⑤应用苏木素伊红(Hematoxylin and eosin, HE)染色观察结肠组织病理学改变并评分；⑥测定结肠组织髓过氧化物酶(myeloperoxidase, MPO)含量；⑦分离肠黏膜固有层单个核细胞(laminapropria mononuclear cells, LPMC)并计数，应用流式细胞术(Flow cytometry,FC)检测Th9(CD4+IL9+T)细胞占CD4+T细胞比例。 结果：①体重改变：Control/WT组与Control/Tg组小鼠体重增加，分别为42.06%±6.41%和35.26%±2.70%；DSS/WT组小鼠体重虽亦有增加，但增加幅度显著低于Control/WT组(9.28%±5.70%vs42.06%±6.41%,P0.05)；DSS/Tg组小鼠体重较原始体重降低，显著低于Control/Tg组(-9.66%±2.50%vs35.26%±1.56%, P0.05)，并且显著低于DSS/WT组(-9.66%±2.50%vs9.28%±5.70%, P0.05)。②DAI评分：Control/WT组和Control/Tg组小鼠DAI评分均为(0.00±0.00)，两组比较无统计学差异；DSS/WT组与DSS/Tg组小鼠的DAI评分逐渐升高，且DSS/Tg组显著高于DSS/WT组(2.55±0.90vs1.58±0.70, P0.05)。③结肠长度、大体形态学及病理学评分：Control/WT组与Control/Tg组结肠形态正常，H＆E染色未见粘膜破损、炎症细胞浸润及溃疡形成，而DSS/WT组与Control/WT组相比，结肠长度显著缩短(6.38±0.47vs7.63±0.54, P0.05)，形态学评分显著增高(1.60±0.31vs0.00±0.00, P0.05)，病理见黏膜上皮缺损、大量炎症细胞浸润、浅溃疡生成，病理评分也显著高于Control/WT组(9.50±0.79vs0.00±0.00, P0.05)；DSS/Tg组小鼠的结肠长度显著短于DSS/WT组(5.30±0.18vs6.38±0.47, P0.05)，结肠形态学评分及病理评分也显著高于DSS/WT组，分别为(2.80±0.64vs1.60±0.31, P0.05)和(11.85±0.86vs9.50±0.79, P0.05)。④MPO活性检测：DSS/WT组的MPO活性显著高于Control/WT组(1.48±0.40vs0.65±0.26, P0.05)；DSS/Tg组的MPO活性显著高于Control/Tg组(2.20±0.45vs0.93±0.25U/g, P0.05)；DSS/Tg组高于DSS/WT组(2.20±0.45vs1.48±0.40, P0.05)，差异无统计学意义。⑤分离、计数LPMC，应用流式细胞术检测LPMC中Th9细胞占CD4+T细胞比例：DSS/WT组的LPMC数显著高于Control/WT组(2.65×106±0.32×106vs1.68×106±0.15×106, P0.05)；DSS/Tg组的LPMC数显著高于Control/Tg组(3.70×106±0.28×106vs1.72×106±0.17×106, P0.05)和DSS/WT组(3.70×106±0.28×106vs2.65×106±0.32×106, P0.05)。结肠组织LPMC中Th9细胞占CD4+T细胞比例：Control/WT组为0.10%±0.02%，Control/Tg组为0.12%±0.01%。DSS/WT组显著高于Control/WT组(0.23%±0.03%vs0.10%±0.02%, P0.05)；DSS/Tg组显著高于Control/Tg组(0.54%±0.04%vs0.12%±0.01%, P0.05)和DSS/WT组(0.54%±0.04%vs0.23%±0.03%,P0.05)。 结论：TL1A可能通过促进Th9细胞活化加重结肠粘膜炎症。
[Abstract]:Ulcerative colitis (ulcerative colitis, UC) is a kind of inflammatory bowel disease (inflammatorybowel disease, IBD). Its pathogenesis is still unknown. It is considered that intestinal mucosal immune abnormalities, continuous intestinal infection, heredity and environment are involved in the development of the disease, and the imbalance of immune regulation is the important pathogenic factor.CD4+T of UC. Cells are the most important effective cells in the immune system, including T helper cell (Th cell) and regulatory T cells (Tregulatory T cell, Treg). It plays an important role in regulating the immune response and maintaining immune balance in the immune response and maintaining the immune balance, which leads to the excessive proliferation of the.CD4+T cells. Differentiation leads to the imbalance between proinflammatory and anti-inflammatory immune systems. 1.
Recent studies have shown that the combination of transforming growth factor beta (transforming growth factor- beta, TGF- beta) and interleukin (interleukin, IL) -4 can induce CD4+ initial T cells to differentiate into a group of new T cell subsets secreting IL-9, and the activated Th2 cells can also produce the same results in the case of isolated beta. Secreting cytokine IL-9 and inducing differentiation pathway are different from other subgroups of T cells, so.Th9 cells named Th9 (CD4+IL-9+) cells are involved in the inflammatory progression of autoimmune cerebrospinal meningitis, tuberculous pleuritis, allergic dermatitis and other inflammatory diseases, and the recombinant activation gene 1 deficiency (recombination-activating gene1-d) is given. Eficient, RAG-1) mouse Th9 cells can also successfully induce colitis in mice.
The tumor necrosis factor like ligand 1A (tumor necrosis factor-like ligand1aberrance, TL1A) is a protein product of the tumor necrosis factor superfamily member 15 (TNF superfamily member15, TNFSF15), mainly expressed in human endothelial cells, including the human umbilical vein endothelial cells, adult skin microvascular endothelial cells and the myometrium of the uterus. The present study shows that TL1A is a susceptible gene for IBD. It can activate Th1, Th2 and Th17 cells by combining with death receptor 3 (death receptor, DR3), causing intestinal mucosal immune disorder and causing intestinal inflammation in IBD. However, the effect of TL1A on Th9 cells is not clear. This study mainly through the detection of TL1A transgenic mice and mice. The level of Th9 cells in colon tissue was investigated to investigate the effect of TL1A on Th9 cell activation.
Objective: To investigate the effect of TL1A on the activation of Th9 cells in colonic tissue of mice with chronic experimental colitis.
Methods: (1) in this experiment, the transgenic mice and WT mice with the lymphocyte overexpression of TL1A were used to identify and screen the TL1A transgenic mice (LCK-CD2-TL1A-GFP, L-Tg), and 12 C57BL/6WT mice with the same genetic background (15~ 18G, 6~8 weeks of age) were randomly divided into Control/WT group, DSS/WT group, each. Group 6, 12 L-Tg mice (body weight 15~18g, 6~8 week age) were randomly divided into group Control/Tg, DSS/Tg group, 6.Control groups in each group drank distilled water, group DSS drank 3% dextran sodium sulfate sodium sulfate (dextran sulfate sodium, DSS), time segments were respectively 1~5 days, day, day, day and day, and the rest of the time drinking distilled Water, twenty-ninth days of death in animals; (3) observe the mental state of the mice, body weight, activity, hair glossiness, stool shape, and stool occult blood, disease activityindex (DAI) score, evaluate the degree of colitis, and observe the morphological changes of the colon, record the length of the colon, and make a gross score; Hematoxylin and eosin (HE) staining was used to observe the pathological changes and score of colonic tissue; (6) the content of myeloperoxidase (myeloperoxidase, MPO) in the colon tissue was measured; and (laminapropria mononuclear cells, LPMC) was separated from the intestinal mucosa of the intestinal mucosa (laminapropria mononuclear cells, LPMC), and the flow cytometry (Flow cytometry) was applied. FC) to detect Th9 (CD4+IL9+T) cells in proportion to CD4+T cells.
Results: (1) weight change: the weight of group Control/WT and group Control/Tg increased, 42.06% + 6.41% and 35.26% + 2.70%, respectively, while the weight of group DSS/WT increased, but the increase was significantly lower than that of group Control/WT (9.28% + 5.70%vs42.06% 6.41%, P0.05). The weight of DSS/Tg group was lower than that of the original group, which was significantly lower than that of the Control/Tg group. (-9.66% + 2.50%vs35.26% + 1.56%, P0.05), and significantly lower than group DSS/WT (-9.66% + 2.50%vs9.28% + 5.70%, P0.05). (2) DAI score: Control/WT group and Control/Tg group mice DAI score (0 + 0), there was no statistical difference between the two groups, and the DSS/WT group and the group of mice increased gradually, and the group was significantly higher than that of the group ( 2.55 + 0.90vs1.58 + 0.70, P0.05). (3) colon length, gross morphology and pathological score: the colonic morphology of group Control/WT and Control/Tg was normal. No mucosa damage, inflammatory cell infiltration and ulcers were found in H & E staining, and the length of colon was significantly shortened (6.38 + 0.47vs7.63 + 0.54, P0.05) in DSS/WT group and Control/WT group. The scores were significantly higher (1.60 + 0.31vs0.00 + 0, P0.05). The pathological changes of mucosa epithelial defect, inflammatory cell infiltration, shallow ulcer formation, and pathological score were significantly higher than that of group Control/WT (9.50 + 0.79vs0.00 + 0, P0.05), and the colon length of group DSS/Tg mice was significantly shorter than that of DSS /WT group (5.30 + 0.18vs6.38 + 0.47, P0.05), and the morphological score of colon and colonic morphology. The pathological score was also significantly higher than that in the DSS/WT group (2.80 + 0.64vs1.60 + 0.31, P0.05) and (11.85 + 0.86vs9.50 + 0.79, P0.05). The activity of MPO in the DSS/WT group was significantly higher than that in the Control/WT group (1.48 + 0.40vs0.65 + 0.26, P0.05), and the activity of the DSS/Tg group was significantly higher than that of the group (2.20 + + 0.26). Group S/Tg was higher than group DSS/WT (2.20 + 0.45vs1.48 + 0.40, P0.05), the difference was not statistically significant. (5) separation, counting LPMC, and using flow cytometry to detect the proportion of CD4+T cells in LPMC: the LPMC number of DSS/WT group was significantly higher than that of Control/WT group (2.65 * 106 + 0.32 * 106vs1.68 * 106 + 0.15 * 106,). Group rol/Tg (3.70 x 106 + 0.28 * 106vs1.72 * 106 + 0.17 x 106, P0.05) and group DSS/WT (3.70 * 106 + 0.28 x 106vs2.65 x 106 + 0.32 x 106, P0.05). The proportion of Th9 cells in the colon tissue LPMC is the proportion of CD4+T cells: Control/WT group is 0.10% + 0.02%, Control/Tg group is significantly higher than that of the group. 05); DSS/Tg group was significantly higher than that of Control/Tg group (0.54% + 0.04%vs0.12% + 0.01%, P0.05) and DSS/WT group (0.54% + 0.04%vs0.23% + 0.03%, P0.05).
Conclusion: TL1A may aggravate colonic mucosal inflammation by promoting Th9 cell activation.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R574.62
本文编号：2146583
[Abstract]:Ulcerative colitis (ulcerative colitis, UC) is a kind of inflammatory bowel disease (inflammatorybowel disease, IBD). Its pathogenesis is still unknown. It is considered that intestinal mucosal immune abnormalities, continuous intestinal infection, heredity and environment are involved in the development of the disease, and the imbalance of immune regulation is the important pathogenic factor.CD4+T of UC. Cells are the most important effective cells in the immune system, including T helper cell (Th cell) and regulatory T cells (Tregulatory T cell, Treg). It plays an important role in regulating the immune response and maintaining immune balance in the immune response and maintaining the immune balance, which leads to the excessive proliferation of the.CD4+T cells. Differentiation leads to the imbalance between proinflammatory and anti-inflammatory immune systems. 1.
Recent studies have shown that the combination of transforming growth factor beta (transforming growth factor- beta, TGF- beta) and interleukin (interleukin, IL) -4 can induce CD4+ initial T cells to differentiate into a group of new T cell subsets secreting IL-9, and the activated Th2 cells can also produce the same results in the case of isolated beta. Secreting cytokine IL-9 and inducing differentiation pathway are different from other subgroups of T cells, so.Th9 cells named Th9 (CD4+IL-9+) cells are involved in the inflammatory progression of autoimmune cerebrospinal meningitis, tuberculous pleuritis, allergic dermatitis and other inflammatory diseases, and the recombinant activation gene 1 deficiency (recombination-activating gene1-d) is given. Eficient, RAG-1) mouse Th9 cells can also successfully induce colitis in mice.
The tumor necrosis factor like ligand 1A (tumor necrosis factor-like ligand1aberrance, TL1A) is a protein product of the tumor necrosis factor superfamily member 15 (TNF superfamily member15, TNFSF15), mainly expressed in human endothelial cells, including the human umbilical vein endothelial cells, adult skin microvascular endothelial cells and the myometrium of the uterus. The present study shows that TL1A is a susceptible gene for IBD. It can activate Th1, Th2 and Th17 cells by combining with death receptor 3 (death receptor, DR3), causing intestinal mucosal immune disorder and causing intestinal inflammation in IBD. However, the effect of TL1A on Th9 cells is not clear. This study mainly through the detection of TL1A transgenic mice and mice. The level of Th9 cells in colon tissue was investigated to investigate the effect of TL1A on Th9 cell activation.
Objective: To investigate the effect of TL1A on the activation of Th9 cells in colonic tissue of mice with chronic experimental colitis.
Methods: (1) in this experiment, the transgenic mice and WT mice with the lymphocyte overexpression of TL1A were used to identify and screen the TL1A transgenic mice (LCK-CD2-TL1A-GFP, L-Tg), and 12 C57BL/6WT mice with the same genetic background (15~ 18G, 6~8 weeks of age) were randomly divided into Control/WT group, DSS/WT group, each. Group 6, 12 L-Tg mice (body weight 15~18g, 6~8 week age) were randomly divided into group Control/Tg, DSS/Tg group, 6.Control groups in each group drank distilled water, group DSS drank 3% dextran sodium sulfate sodium sulfate (dextran sulfate sodium, DSS), time segments were respectively 1~5 days, day, day, day and day, and the rest of the time drinking distilled Water, twenty-ninth days of death in animals; (3) observe the mental state of the mice, body weight, activity, hair glossiness, stool shape, and stool occult blood, disease activityindex (DAI) score, evaluate the degree of colitis, and observe the morphological changes of the colon, record the length of the colon, and make a gross score; Hematoxylin and eosin (HE) staining was used to observe the pathological changes and score of colonic tissue; (6) the content of myeloperoxidase (myeloperoxidase, MPO) in the colon tissue was measured; and (laminapropria mononuclear cells, LPMC) was separated from the intestinal mucosa of the intestinal mucosa (laminapropria mononuclear cells, LPMC), and the flow cytometry (Flow cytometry) was applied. FC) to detect Th9 (CD4+IL9+T) cells in proportion to CD4+T cells.
Results: (1) weight change: the weight of group Control/WT and group Control/Tg increased, 42.06% + 6.41% and 35.26% + 2.70%, respectively, while the weight of group DSS/WT increased, but the increase was significantly lower than that of group Control/WT (9.28% + 5.70%vs42.06% 6.41%, P0.05). The weight of DSS/Tg group was lower than that of the original group, which was significantly lower than that of the Control/Tg group. (-9.66% + 2.50%vs35.26% + 1.56%, P0.05), and significantly lower than group DSS/WT (-9.66% + 2.50%vs9.28% + 5.70%, P0.05). (2) DAI score: Control/WT group and Control/Tg group mice DAI score (0 + 0), there was no statistical difference between the two groups, and the DSS/WT group and the group of mice increased gradually, and the group was significantly higher than that of the group ( 2.55 + 0.90vs1.58 + 0.70, P0.05). (3) colon length, gross morphology and pathological score: the colonic morphology of group Control/WT and Control/Tg was normal. No mucosa damage, inflammatory cell infiltration and ulcers were found in H & E staining, and the length of colon was significantly shortened (6.38 + 0.47vs7.63 + 0.54, P0.05) in DSS/WT group and Control/WT group. The scores were significantly higher (1.60 + 0.31vs0.00 + 0, P0.05). The pathological changes of mucosa epithelial defect, inflammatory cell infiltration, shallow ulcer formation, and pathological score were significantly higher than that of group Control/WT (9.50 + 0.79vs0.00 + 0, P0.05), and the colon length of group DSS/Tg mice was significantly shorter than that of DSS /WT group (5.30 + 0.18vs6.38 + 0.47, P0.05), and the morphological score of colon and colonic morphology. The pathological score was also significantly higher than that in the DSS/WT group (2.80 + 0.64vs1.60 + 0.31, P0.05) and (11.85 + 0.86vs9.50 + 0.79, P0.05). The activity of MPO in the DSS/WT group was significantly higher than that in the Control/WT group (1.48 + 0.40vs0.65 + 0.26, P0.05), and the activity of the DSS/Tg group was significantly higher than that of the group (2.20 + + 0.26). Group S/Tg was higher than group DSS/WT (2.20 + 0.45vs1.48 + 0.40, P0.05), the difference was not statistically significant. (5) separation, counting LPMC, and using flow cytometry to detect the proportion of CD4+T cells in LPMC: the LPMC number of DSS/WT group was significantly higher than that of Control/WT group (2.65 * 106 + 0.32 * 106vs1.68 * 106 + 0.15 * 106,). Group rol/Tg (3.70 x 106 + 0.28 * 106vs1.72 * 106 + 0.17 x 106, P0.05) and group DSS/WT (3.70 * 106 + 0.28 x 106vs2.65 x 106 + 0.32 x 106, P0.05). The proportion of Th9 cells in the colon tissue LPMC is the proportion of CD4+T cells: Control/WT group is 0.10% + 0.02%, Control/Tg group is significantly higher than that of the group. 05); DSS/Tg group was significantly higher than that of Control/Tg group (0.54% + 0.04%vs0.12% + 0.01%, P0.05) and DSS/WT group (0.54% + 0.04%vs0.23% + 0.03%, P0.05).
Conclusion: TL1A may aggravate colonic mucosal inflammation by promoting Th9 cell activation.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R574.62
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