TL1A對慢性實驗性結(jié)腸炎小鼠結(jié)腸組織Th9細(xì)胞活化的影響
發(fā)布時間：2018-07-26 16:23
【摘要】：潰瘍性結(jié)腸炎(ulcerative colitis, UC)是炎癥性腸病(inflammatorybowel disease, IBD)的一種，其發(fā)病機(jī)制尚不明了。目前認(rèn)為腸黏膜免疫異常、持續(xù)腸道感染、遺傳和環(huán)境等因素均參與了該病的發(fā)生發(fā)展，其中免疫調(diào)控失衡是UC的重要致病因素。CD4+T細(xì)胞是免疫系統(tǒng)中最重要的效能細(xì)胞，包括輔助性T細(xì)胞(T helper cell, Th cell)和調(diào)節(jié)性T細(xì)胞(Tregulatory T cell, Treg)，在調(diào)控體內(nèi)免疫應(yīng)答和維持免疫平衡中發(fā)揮重要作用。CD4+T細(xì)胞過度增殖、分化導(dǎo)致促炎與抗炎免疫失衡是UC的主要特點之一。 近年來的研究表明，轉(zhuǎn)化生長因子β(transforming growth factor-β,TGF-β)和白細(xì)胞介素(interleukin, IL)-4聯(lián)合可誘導(dǎo)CD4+初始T細(xì)胞分化成一群大量分泌IL-9的新T細(xì)胞亞群，活化的Th2細(xì)胞在TGF-β單獨存在的情況下也可以產(chǎn)生相同的結(jié)果。因其主要分泌細(xì)胞因子IL-9，且誘導(dǎo)分化途徑又不同于T細(xì)胞的其他亞群，所以被命名為Th9(CD4+IL-9+)細(xì)胞。Th9細(xì)胞參與了自身免疫性腦脊髓膜炎、結(jié)核性胸膜炎、過敏性皮炎等多種炎性疾病的炎癥進(jìn)展，給重組活化基因1缺陷(recombination-activating gene1-deficient, RAG-1)小鼠回輸Th9細(xì)胞亦可成功誘導(dǎo)小鼠結(jié)腸炎癥。 腫瘤壞死因子樣配體1A(tumor necrosis factor-like ligand1aberrance,TL1A)是腫瘤壞死因子超家族成員15(TNF superfamily member15,TNFSF15)基因的蛋白產(chǎn)物，主要表達(dá)在人類的內(nèi)皮細(xì)胞，包括人臍靜脈內(nèi)皮細(xì)胞、成人皮膚微血管內(nèi)皮細(xì)胞和子宮肌層內(nèi)皮細(xì)胞等。目前研究表明，TL1A是IBD的易感基因，可通過與死亡受體3(death receptor, DR3)結(jié)合，激活Th1、Th2和Th17細(xì)胞，致腸黏膜免疫紊亂，引發(fā)IBD的腸道炎癥反應(yīng)。然而目前TL1A對Th9細(xì)胞的影響尚不清楚。本研究主要是通過檢測TL1A轉(zhuǎn)基因小鼠及WT小鼠結(jié)腸組織中Th9細(xì)胞水平探討TL1A對Th9細(xì)胞活化的影響。 目的：探討TL1A對慢性實驗性結(jié)腸炎小鼠結(jié)腸組織中Th9細(xì)胞活化的影響。 方法：①本實驗以淋巴細(xì)胞過表達(dá)TL1A的轉(zhuǎn)基因小鼠及WT小鼠為研究對象，采用RT-PCR方法進(jìn)行TL1A的轉(zhuǎn)基因(LCK-CD2-TL1A-GFP,L-Tg)小鼠的鑒定與篩選；②12只具有相同遺傳背景的C57BL/6WT小鼠(體重15~18g，6~8周齡)隨機(jī)分為Control/WT組、DSS/WT組，每組6只；12只L-Tg小鼠(體重15~18g，6~8周齡)隨機(jī)分為Control/Tg組、DSS/Tg組，每組6只。Control組飲用蒸餾水，DSS組間斷飲用3%右旋葡聚糖硫酸鈉(dextran sulfate sodium, DSS)，時間段分別為第1~5天、第8~12天、第15~19天、第22~26天及第27、28天，其余時間飲用蒸餾水，第29天處死動物；③每日觀察小鼠的精神狀態(tài)、體重、活動情況、毛發(fā)光澤度、糞便形狀、大便潛血情況等，進(jìn)行疾病活動指數(shù)(disease activityindex, DAI)評分，評估結(jié)腸炎癥程度；④觀察結(jié)腸形態(tài)學(xué)改變，記錄結(jié)腸長度變化，，進(jìn)行大體評分；⑤應(yīng)用蘇木素伊紅(Hematoxylin and eosin, HE)染色觀察結(jié)腸組織病理學(xué)改變并評分；⑥測定結(jié)腸組織髓過氧化物酶(myeloperoxidase, MPO)含量；⑦分離腸黏膜固有層單個核細(xì)胞(laminapropria mononuclear cells, LPMC)并計數(shù)，應(yīng)用流式細(xì)胞術(shù)(Flow cytometry,FC)檢測Th9(CD4+IL9+T)細(xì)胞占CD4+T細(xì)胞比例。 結(jié)果：①體重改變：Control/WT組與Control/Tg組小鼠體重增加，分別為42.06%±6.41%和35.26%±2.70%；DSS/WT組小鼠體重雖亦有增加，但增加幅度顯著低于Control/WT組(9.28%±5.70%vs42.06%±6.41%,P0.05)；DSS/Tg組小鼠體重較原始體重降低，顯著低于Control/Tg組(-9.66%±2.50%vs35.26%±1.56%, P0.05)，并且顯著低于DSS/WT組(-9.66%±2.50%vs9.28%±5.70%, P0.05)。②DAI評分：Control/WT組和Control/Tg組小鼠DAI評分均為(0.00±0.00)，兩組比較無統(tǒng)計學(xué)差異；DSS/WT組與DSS/Tg組小鼠的DAI評分逐漸升高，且DSS/Tg組顯著高于DSS/WT組(2.55±0.90vs1.58±0.70, P0.05)。③結(jié)腸長度、大體形態(tài)學(xué)及病理學(xué)評分：Control/WT組與Control/Tg組結(jié)腸形態(tài)正常，H＆E染色未見粘膜破損、炎癥細(xì)胞浸潤及潰瘍形成，而DSS/WT組與Control/WT組相比，結(jié)腸長度顯著縮短(6.38±0.47vs7.63±0.54, P0.05)，形態(tài)學(xué)評分顯著增高(1.60±0.31vs0.00±0.00, P0.05)，病理見黏膜上皮缺損、大量炎癥細(xì)胞浸潤、淺潰瘍生成，病理評分也顯著高于Control/WT組(9.50±0.79vs0.00±0.00, P0.05)；DSS/Tg組小鼠的結(jié)腸長度顯著短于DSS/WT組(5.30±0.18vs6.38±0.47, P0.05)，結(jié)腸形態(tài)學(xué)評分及病理評分也顯著高于DSS/WT組，分別為(2.80±0.64vs1.60±0.31, P0.05)和(11.85±0.86vs9.50±0.79, P0.05)。④MPO活性檢測：DSS/WT組的MPO活性顯著高于Control/WT組(1.48±0.40vs0.65±0.26, P0.05)；DSS/Tg組的MPO活性顯著高于Control/Tg組(2.20±0.45vs0.93±0.25U/g, P0.05)；DSS/Tg組高于DSS/WT組(2.20±0.45vs1.48±0.40, P0.05)，差異無統(tǒng)計學(xué)意義。⑤分離、計數(shù)LPMC，應(yīng)用流式細(xì)胞術(shù)檢測LPMC中Th9細(xì)胞占CD4+T細(xì)胞比例：DSS/WT組的LPMC數(shù)顯著高于Control/WT組(2.65×106±0.32×106vs1.68×106±0.15×106, P0.05)；DSS/Tg組的LPMC數(shù)顯著高于Control/Tg組(3.70×106±0.28×106vs1.72×106±0.17×106, P0.05)和DSS/WT組(3.70×106±0.28×106vs2.65×106±0.32×106, P0.05)。結(jié)腸組織LPMC中Th9細(xì)胞占CD4+T細(xì)胞比例：Control/WT組為0.10%±0.02%，Control/Tg組為0.12%±0.01%。DSS/WT組顯著高于Control/WT組(0.23%±0.03%vs0.10%±0.02%, P0.05)；DSS/Tg組顯著高于Control/Tg組(0.54%±0.04%vs0.12%±0.01%, P0.05)和DSS/WT組(0.54%±0.04%vs0.23%±0.03%,P0.05)。 結(jié)論：TL1A可能通過促進(jìn)Th9細(xì)胞活化加重結(jié)腸粘膜炎癥。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R574.62
本文編號：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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R574.62
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