【作者】 王冬；

【导师】 张晓岚；

【作者基本信息】 河北医科大学 ， 内科学， 2017， 博士

【摘要】 炎症性肠病(Inflammatory bowel disease,IBD)包括溃疡性结肠炎(Ulcerative colitis,UC)和克罗恩病(Crohn’s disease,CD),发病率呈逐年上升趋势。其发病机制尚不十分明确,目前认为肠黏膜免疫异常、持续肠道感染、遗传和环境等因素共同参与IBD的发生发展。由于具有慢性及反复发作的特点,严重影响患者生活质量,给临床治疗带来极大的挑战。因此,进一步探讨和阐明IBD的发病机制对于寻找新的治疗方法具有重要的意义。肠黏膜免疫异常在IBD发病中发挥着重要作用,而CD4+T细胞是免疫系统中最重要的效能细胞。Th9细胞是新发现的一种CD4+T细胞亚群,主要通过分泌IL-9发挥促炎作用。Th9细胞的特异性转录因子为ETS(E26-transformation specific)家族转录因子富含嘌呤盒1(Purine-rich box1,PU.1)。TGF-β和IL-4是诱导Th9细胞产生和分化的关键细胞因子。体外在TGF-β和IL-4及IL-2等联合刺激下CD4 Na?ve T细胞可被诱导分化成为Th9细胞。Th9细胞参与多种自身免疫性疾病的发生,亦有少数报道发现其可促进IBD进程。多项研究发现,UC患者血清与结肠组织中Th9细胞与IL-9水平明显升高。在动物实验中也发现在实验性结肠炎小鼠的血清、肠系膜淋巴结、脾脏和结肠组织中Th9细胞的数量及IL-9表达均明显升高,说明Th9细胞与IL-9在结肠炎的发病中起到重要的作用。TL1A是新近发现的一种肿瘤坏死因子家族新成员。GWAS研究证实TNFSF15是IBD的易感基因。TL1A可为T淋巴细胞的激活提供协同刺激信号,刺激淋巴细胞增殖及细胞因子产生,进而在哮喘、类风湿性关节炎和IBD等多种疾病中发挥重要作用。研究发现,TL1A可通过与其受体结合协同刺激IL-12/IL-18,介导Th1型免疫反应促进T细胞和NK细胞的IFN-γ分泌,并且可作用于Th17细胞使其分泌IL-17增加。TL1A通过Th1、Th2和Th17细胞在肠道炎症中的作用已取得进展,但是仅有少数研究报道了在过敏性肺炎小鼠模型中TL1A可促进Th9细胞分化和IL-9生成。目前尚无TL1A与Th9细胞联合作用在IBD中的研究。TL1A是否通过Th9细胞及其分泌因子IL-9来调控肠道炎症尚不清楚。因此,本实验拟采用淋系细胞过表达TL1A的转基因小鼠及具有相同遗传背景的野生型小鼠为研究对象,建立DSS诱导的慢性实验性结肠炎模型,造模成功后给予小鼠腹腔注射IL-9抗体,并分离脾脏和肠系膜淋巴结单个核细胞,通过磁珠分选CD4 Na?ve T细胞,体外诱导分化成为Th9细胞,应用TL1A刺激,再分别加入TL1A抗体和IL-9抗体进行干预,在动物和细胞水平探讨TL1A在慢性实验性结肠炎中对Th9细胞和IL-9的作用与机制及IL-9抗体对慢性实验性结肠炎的治疗作用,以期为IBD的防治提供新的理论依据。具体实验分为以下三部分:第一部分:Th9细胞和IL-9在TL1A过表达慢性实验性结肠炎中的表达变化目的:探讨TL1A对慢性实验性结肠炎小鼠中Th9细胞及IL-9的作用与机制方法:1)PCR方法扩增TL1A DNA片段,琼脂糖凝胶电泳检测进行LCK-CD2-TL1A-GFP-transgenic小鼠的鉴定。2)通过饮用葡聚糖硫酸钠(Dextran sulfate sodium,DSS)28天成功建立慢性实验性结肠炎模型:将Tg小鼠与有相同遗传背景的C57BL/6 WT小鼠(6~8周)随机分为:(1)Control/WT组;(2)DSS/WT组;(3)Control/Tg组;(4)DSS/Tg组,每组10只。小鼠分别于第1~5天、8~12天、15~19天和22~26天饮用2.5%DSS,其他时间饮用蒸馏水。共4个循环,每个循环的第1、3、5天进行疾病活动指数(Disease activity index,DAI)评分,第29天处死动物。3)炎症改变的评估:包括体重(Body weight,BW)改变、DAI、结肠长度、结肠形态学改变、病理组织学评分和MPO活性。4)免疫荧光共染方法检测CD4+IL-9+T细胞比例;流式细胞仪(Flow cytometry instrument,FCM)检测脾脏、MLN和肠黏膜固有层分离的单个核细胞中CD4+IL-9+T细胞比例;Real time PCR检测IL-9 mRNA在结肠组织中的表达;ELISA方法检测血清中和脾脏、MLN、肠黏膜固有层分离的单个核细胞培养上清液中IL-9的分泌。5)Real time PCR检测慢性实验性结肠炎小鼠结肠组织中TGF-β和IL-4 mRNA的表达;ELISA方法检测血液中和脾脏、MLN、肠黏膜固有层单个核细胞培养上清液中TGF-β和IL-4分泌。6)Western blot检测慢性实验性结肠炎小鼠结肠组织中TL1A和PU.1的蛋白定量表达;Real time PCR检测慢性实验性结肠炎小鼠结肠组织中TL1A mRNA和PU.1 mRNA的表达情况。结果:1)LCK-CD2-TL1A-GFP特定引物扩增小鼠DNA,凝胶成像显示Tg小鼠在192 bp处可见较强的荧光信号,据此进行转基因小鼠的鉴定。2)成功建立慢性结肠炎小鼠模型:DSS/WT组和DSS/Tg组小鼠在饮用2.5%DSS后,与Control/WT组和Control/Tg组小鼠相比体重明显下降,DAI评分明显升高,结肠黏膜充血、水肿、肠壁增厚,结肠大体形态学评分明显升高,结肠长度缩短;与DSS/WT组小鼠相比,DSS/Tg组小鼠结肠炎症更严重(P<0.05)。3)病理评分:DSS/WT组和DSS/Tg组小鼠出现了结肠黏膜缺损,腺体破坏或消失,杯状细胞减少,并可见黏膜、黏膜下层甚至肌层大量淋巴细胞浸润,与相应Control/WT组和Control/Tg组小鼠相比,DSS/WT组和DSS/Tg组小鼠病理评分明显增高(8.25±0.50 vs0.00±0.00,P<0.05)和(12.50±0.58 vs 0.00±0.00,P<0.05);与DSS/WT组小鼠相比,DSS/Tg组小鼠病理评分增加更为明显(12.50±0.58 vs 8.25±0.50,P<0.05)。4)MPO活性:与Control/WT组小鼠相比,DSS/WT组小鼠结肠MPO活性明显升高(1.09±0.21 vs 0.43±0.11,P<0.05)。与Control/Tg组小鼠相比,DSS/Tg组小鼠结肠MPO活性明显增加(1.80±0.12 vs 0.60±0.07,P<0.05)。DSS/Tg组小鼠较DSS/WT组小鼠结肠MPO活性增加程度更大(1.80±0.12 vs 1.09±0.21,P<0.05)。5)免疫荧光方法检测结肠组织中CD4+IL-9+T细胞比例:与Control/WT组小鼠相比,DSS/WT组小鼠结肠组织中CD4+IL-9+平均光密度值显著升高(0.24±0.03 vs 0.09±0.01,P<0.05);同样,与Control/Tg组小鼠相比,DSS/Tg组小鼠结肠组织中CD4+IL-9+平均光密度值也显著升高(0.44±0.03 vs 0.10±0.01,P<0.05);与DSS/WT组小鼠相比,DSS/Tg组小鼠结肠组织中CD4+IL-9+平均光密度值升高程度更大(0.44±0.03 vs 0.24±0.03,P<0.05)。FCM检测脾脏、MLN和肠黏膜固有层单个核细胞CD4+IL-9+T细胞比例,均可见DSS/Tg组较DSS/WT组单个核细胞CD4+IL-9+T细胞比例增加(脾脏单个核细胞CD4+IL-9+T细胞比例:2.42%±0.37%vs 1.29%±0.24%,P<0.05;MLN单个核细胞CD4+IL-9+T细胞比例:2.53%±0.38%vs 1.76%±0.43%,P<0.05;肠黏膜固有层单个核细胞CD4+IL-9+T细胞比例:4.42%±0.45%vs 2.67%±0.58%,P<0.05)。Real time PCR检测结肠组织中IL-9 mRNA的表达:与Control/WT组小鼠相比,DSS/WT组小鼠结肠组织中IL-9 mRNA的表达显著升高(3.77±0.68 vs 1.03±0.23,P<0.05);同样,与Control/Tg组小鼠相比,DSS/Tg组小鼠结肠组织中IL-9 mRNA的表达也显著升高(6.19±0.74 vs 1.16±0.33,P<0.05);与DSS/WT组小鼠相比,DSS/Tg组小鼠结肠组织中IL-9 mRNA的表达升高程度更大(6.19±0.74 vs 3.77±0.68,P<0.05)。ELISA方法检测血清中和脾脏、MLN、肠黏膜固有层分离的单个核细胞培养上清液中IL-9分泌的水平,均显示与DSS/WT组小鼠相比,DSS/Tg组小鼠IL-9分泌增加程度更大(血清IL-9水平:380.14pg/m L±15.30 pg/m L vs 253.72 pg/m L±19.97 pg/m L,P<0.05;脾脏单个核细胞培养上清液中IL-9分泌水平:385.02 pg/m L±20.02 pg/m L vs 228.61pg/m L±13.40 pg/m L,P<0.05;MLN单个核细胞培养上清液中IL-9分泌水平:401.74 pg/m L±13.18 pg/m L vs 241.93 pg/m L±17.19 pg/m L,P<0.05;肠黏膜固有层单个核细胞培养上清液中IL-9分泌水平:397.41 pg/m L±12.62pg/m L vs 248.43 pg/m L±20.62 pg/m L,P<0.05)。6)Real time PCR检测慢性实验性结肠炎小鼠结肠组织中TGF-β和IL-4 mRNA的表达:Control/WT组小鼠结肠组织中TGF-β和IL-4 mRNA分别为(1.02±0.20,1.01±0.17),而DSS/WT组小鼠结肠组织中TGF-β和IL-4 mRNA分别为(6.22±0.53,3.35±0.57),与Control/WT组相比表达明显升高(P<0.05)。Control/Tg组小鼠结肠组织中TGF-β和IL-4 mRNA分别为(1.16±0.24,1.29±0.33),而DSS/Tg组小鼠结肠组织中TGF-β和IL-4 mRNA分别为(15.06±1.63,4.80±0.89),与Control/Tg组相比表达明显升高(P<0.05)。与DSS/WT组小鼠结肠组织中TGF-β和IL-4 mRNA表达(6.22±0.53,3.35±0.57)相比,DSS/Tg组小鼠表达(15.06±1.63,4.80±0.89)进一步升高(P<0.05);ELISA方法分别检测脾脏、MLN和肠黏膜固有层单个核细胞培养上清液中TGF-β和IL-4分泌:DSS/Tg组TGF-β和IL-4分泌水平较DSS/WT组明显升高(脾脏单个核细胞培养上清液中TGF-β分泌水平:514.69 pg/m L±47.13pg/m L vs 280.66 pg/m L±26.84 pg/m L,P<0.05;脾脏单个核细胞培养上清液中IL-4分泌水平:160.49 pg/m L±9.20 pg/m L vs 117.99 pg/m L±8.87pg/m L,P<0.05;MLN单个核细胞培养上清液中TGF-β分泌水平:558.93pg/m L±30.10 pg/m L vs 305.71 pg/m L±29.32 pg/m L,P<0.05;MLN单个核细胞培养上清液中IL-4分泌水平:169.39 pg/m L±10.42 pg/m L vs 115.38pg/m L±5.80 pg/m L,P<0.05;肠黏膜固有层单个核细胞培养上清液中TGF-β分泌水平:622.93 pg/m L±34.76 pg/m L vs 319.62 pg/m L±39.45 pg/m L,P<0.05;肠黏膜固有层单个核细胞培养上清液中IL-4分泌水平:175.92pg/m L±8.27 pg/m L vs 123.83 pg/m L±6.15 pg/m L,P<0.05)。7)Real time PCR检测慢性实验性结肠炎小鼠结肠组织中TL1A和PU.1 mRNA的表达:Control/WT组小鼠结肠组织TL1A和PU.1 mRNA分别为(1.01±0.14,1.03±0.26),而DSS/WT组小鼠结肠组织TL1A和PU.1 mRNA分别为(9.19±1.24,2.68±0.46),与Control/WT组相比明显升高(P<0.05);Control/Tg组小鼠结肠组织TL1A和PU.1 mRNA分别为(1.21±0.16,1.20±0.18),而DSS/Tg组小鼠结肠组织TL1A和PU.1 mRNA分别为(12.62±2.01,4.90±0.87),与Control/Tg组相比明显升高(P<0.05);与DSS/WT组小鼠小鼠结肠组织TL1A和PU.1 mRNA(9.19±1.24,2.68±0.46)相比,DSS/Tg组小鼠(12.62±2.01,4.90±0.87)进一步升高(P<0.05)。8)Western blot检测慢性实验性结肠炎小鼠结肠组织中TL1A和PU.1的蛋白表达:Control/WT组小鼠结肠组织TL1A和PU.1蛋白表达分别为(0.16±0.06,0.16±0.04),而DSS/WT组小鼠分别为(0.49±0.10,0.54±0.06),与Control/WT组相比明显升高(P<0.05);Control/Tg组小鼠结肠组织TL1A和PU.1蛋白表达分别为(0.20±0.09,0.20±0.04),而DSS/Tg组小鼠分别为(0.78±0.11,0.76±0.07),与Control/Tg组相比明显升高(P<0.05);与DSS/WT组小鼠小鼠结肠组织TL1A和PU.1蛋白表达(0.49±0.10,0.54±0.06)相比,DSS/Tg组小鼠(0.78±0.11,0.76±0.07)进一步升高(P<0.05)。第二部分:TL1A对Th9细胞的诱导分化作用与机制研究目的:应用CD4 Na?ve T细胞诱导分化为Th9细胞,应用TL1A刺激,再分别加入TL1A抗体和IL-9抗体进行干预,探讨TL1A对Th9细胞的诱导分化作用。方法:1)应用CD4 Na?ve T细胞磁珠分选分别从TL1A过表达Tg小鼠和WT小鼠脾脏与MLN的单个核细胞中阴性分选CD4 Na?ve T细胞,应用FCM鉴定其中CD3~+CD4~+CD44-CD62L+细胞(即CD4 Na?ve T细胞)分选率。2)诱导分化Th9细胞成功后,应用FCM分别检测脾脏和MLN中提取并诱导分化的Th9细胞中CD4+IL-9+T细胞比例。3)在诱导分化Th9细胞过程中加入100 ng/m L TL1A,应用流式细胞仪检测CD4+IL-9+T细胞比例、ELISA方法检测各组培养液上清IL-9分泌水平以及Western blot检测Th9细胞中PU.1的蛋白表达。4)在诱导分化Th9细胞过程中进一步加入4 ug/m L TL1A抗体,应用流式细胞仪检测CD4+IL-9+T细胞比例、ELISA方法检测各组培养液上清IL-9分泌水平以及Western blot检测Th9细胞中PU.1的蛋白表达。5)在诱导分化Th9细胞过程中进一步加入50 ng/m L IL-9抗体,应用流式细胞仪检测CD4+IL-9+T细胞比例、ELISA方法检测各组培养液上清IL-9分泌水平以及Western blot检测Th9细胞中PU.1的蛋白表达。结果:1)应用CD4 Na?ve T细胞磁珠分选试剂盒分别从脾脏和MLN的单个核细胞中阴性分选得到CD4 Na?ve T细胞后,FCM鉴定其中CD3~+CD4~+CD44-CD62L+细胞(即CD4 Na?ve T细胞)分选率,脾脏和MLN分选纯度分别为(90.18%±2.62%)和(92.12%±2.82%),能够满足后续实验要求。2)应用FCM分别检测Th9细胞、脾脏和MLN单个核细胞中CD4+IL-9+T细胞比例:与脾脏单个核细胞相比,脾脏中提取并诱导分化的Th9细胞CD4+IL-9+T细胞比例明显升高(WT组:20.93%±3.60%vs 0.28%±0.10%,P<0.05;Tg组:31.40%±3.66%vs 0.42%±0.10%,P<0.05),且Th9细胞/Tg较Th9细胞/WT组中CD4+IL-9+T细胞比例更高(P<0.05)。同样与MLN单个核细胞相比,MLN中提取并诱导分化的Th9细胞CD4+IL-9+T细胞比例明显升高(WT组:20.50%±3.62%vs 0.31%±0.12%,P<0.05;Tg组:30.53%±3.80%vs 0.42%±0.13%,P<0.05),且Th9细胞/Tg较Th9细胞/WT组中CD4+IL-9+T细胞比例更高(P<0.05)。3)在诱导分化Th9细胞过程中加入TL1A,检测CD4+IL-9+T细胞比例、IL-9分泌水平以及PU.1的蛋白表达:脾脏提取并诱导分化的Th9细胞中,TL1A/WT组(CD4+IL-9+T细胞:30.63%±2.32%vs 21.07%±2.35%,P<0.05;IL-9:3867.63 pg/m L±88.11pg/m L vs 2464.15 pg/m L±122.63 pg/m L,P<0.05;PU.1:1.25±0.08 vs 0.95±0.06,P<0.05)和TL1A/Tg组(CD4+IL-9+T细胞:37.63%±2.26%vs29.90%±2.33%,P<0.05;IL-9:4221.90 pg/m L±81.11 pg/m L vs 2855.90pg/m L±128.59 pg/m L,P<0.05;PU.1:1.42±0.07 vs 1.18±0.06,P<0.05)的CD4+IL-9+T细胞比例、IL-9分泌水平以及PU.1蛋白表达均比各自Control组明显上升,且TL1A/Tg组较TL1A/WT组升高更为明显(P<0.05)。同样MLN提取并诱导分化的Th9细胞中,和各自相应的Control组相比,TL1A/WT组(CD4+IL-9+T细胞:29.80%±2.3%vs 24.60%±1.67%,P<0.05;IL-9:3939.17 pg/m L±166.15 pg/m L vs 2603.82 pg/m L±98.20 pg/m L,P<0.05;PU.1:1.23±0.06 vs 0.93±0.06,P<0.05)和TL1A/Tg组(CD4+IL-9+T细胞:42.67%±2.56%vs 31.40%±2.75%,P<0.05;IL-9:4337.73pg/m L±161.69 pg/m L vs 3142.04 pg/m L±153.75 pg/m L,P<0.05;PU.1:1.41±0.08 vs 1.22±0.08,P<0.05)的CD4+IL-9+T细胞比例、IL-9分泌水平以及PU.1蛋白表达均明显上升,且TL1A/Tg组较TL1A/WT组升高更为明显(P<0.05)。4)在诱导分化Th9细胞过程中加入TL1A抗体,检测CD4+IL-9+T细胞比例、IL-9分泌水平以及PU.1的蛋白表达:脾脏提取并诱导分化的Th9细胞中,TL1A Ab/WT组(CD4+IL-9+T细胞:18.30%±1.51%vs30.63%±2.32%,P<0.05;IL-9:2556.13 pg/m L±83.65 pg/m L;PU.1:0.63±0.07 vs 1.25±0.08,P<0.05)和TL1A Ab/Tg组(CD4+IL-9+T细胞:23.67%±2.06%vs 37.63%±2.26%,P<0.05;IL-9:2556.13 pg/m L±83.65 pg/m L vs3867.63 pg/m L±88.11 pg/m L,P<0.05;PU.1:0.85±0.07 vs 1.42±0.07,P<0.05)的CD4+IL-9+T细胞比例、IL-9分泌水平以及PU.1蛋白表达均比各自TL1A组明显下降,且TL1A Ab/WT组较TL1A Ab/Tg组下降更为明显(P<0.05)。同样MLN提取并诱导分化的Th9细胞中,和各自相应的TL1A组相比,TL1A Ab/WT组(CD4+IL-9+T细胞:17.33%±1.96%vs 29.80%±2.30%,P<0.05;IL-9:2576.57 pg/m L±53.10 pg/m L vs 3939.17 pg/m L±166.15pg/m L,P<0.05;PU.1:0.63±0.09 vs 1.23±0.06,P<0.05)和TL1A Ab/Tg组(CD4+IL-9+T细胞:29.50%±2.01%vs 42.27%±2.56%,P<0.05;IL-9:3111.39 pg/m L±62.44 pg/m L vs 4337.73 pg/m L±161.69 pg/m L,P<0.05;PU.1:0.92±0.10 vs 1.41±0.08,P<0.05)的CD4+IL-9+T细胞比例、IL-9分泌水平以及PU.1蛋白表达均明显下降(P<0.05),且TL1A Ab/WT组较TL1A Ab/Tg组下降更为明显(P<0.05)。5)在诱导分化Th9细胞过程中加入IL-9抗体,检测CD4+IL-9+T细胞比例、IL-9分泌水平以及PU.1的蛋白表达:脾脏提取并诱导分化的Th9细胞中,IL-9 Ab/WT组(CD4+IL-9+T细胞:23.50%±1.93%vs 30.63%±2.32%,P<0.05;IL-9:2218.88pg/m L±90.83 pg/m L vs 3867.63 pg/m L±88.11 pg/m L,P<0.05;PU.1:0.76±0.08 vs 1.25±0.08,P<0.05)和IL-9 Ab/Tg组(CD4+IL-9+T细胞:26.77%±1.90%vs 37.63%±2.26%,P<0.05;IL-9:2669.20 pg/m L±100.65 pg/m L vs4221.90 pg/m L±81.11 pg/m L,P<0.05;PU.1:0.94±0.06 vs 1.42±0.07,P<0.05)的CD4+IL-9+T细胞比例、IL-9分泌水平以及PU.1蛋白表达均比各自TL1A组明显下降(P<0.05),且IL-9 Ab/WT组较IL-9 Ab/Tg组下降更为明显(P<0.05)。同样MLN提取并诱导分化的Th9细胞中,和各自相应的TL1A组相比,IL-9 Ab/WT组(CD4+IL-9+T细胞:24.70%±2.10%vs29.80%±2.30%,P<0.05;IL-9:2215.48 pg/m L±129.40 pg/m L vs 3939.17pg/m L±166.15 pg/m L,P<0.05;PU.1:0.66±0.07 vs 1.23±0.06,P<0.05)和IL-9 Ab/Tg组(CD4+IL-9+T细胞:30.20%±2.40%vs 42.27%±2.56%,P<0.05;IL-9:2719.64 pg/m L±61.32 pg/m L vs 4337.73 pg/m L±161.69 pg/m L,P<0.05;PU.1:0.88±0.08 vs 1.41±0.08,P<0.05)的CD4+IL-9+T细胞比例、IL-9分泌水平以及PU.1蛋白表达均明显下降(P<0.05),且IL-9 Ab/WT组较IL-9 Ab/Tg组下降更为明显(P<0.05)。第三部分:IL-9抗体对TL1A过表达慢性实验性结肠炎的治疗作用目的:研究IL-9抗体对TL1A过表达慢性实验性结肠炎的影响,结合第一、二部分结果进一步证实TL1A对Th9细胞及IL-9的作用,以期为探寻IBD新的治疗方法提供理论依据。方法:1)将Tg C57BL/6小鼠与有相同遗传背景的WT小鼠(体重20~22g,8~10周)随机分为:(1)Control/WT组;(2)DSS/WT组;(3)Isotype Ab/WT组;(4)IL-9 Ab/WT组;(5)Control/Tg;(6)DSS/Tg组;(7)Isotype Ab/Tg组;(8)IL-9 Ab/Tg组,每组10只。Control组饮用蒸馏水;DSS组、IL-9 Ab组和Isotype Ab组小鼠饮用2.5%DSS的时间段为分别为1~5天、8~12天、15~19天和22~26天,27和28天及其他时间饮蒸馏水;IL-9 Ab组和Isotype Ab组第15天开始分别给予IL-9抗体和同型免疫球蛋白G腹腔注射,抗体剂量为100 ug/只,隔日1次。分别于每个循环的第1、3、5天进行DAI评分,第29天处死动物。2)炎症改变的评估:包括体重(Body weight,BW)改变、疾病活动指数(Disease activity index,DAI)、结肠长度、结肠形态学改变、病理评分和MPO活性。3)免疫荧光共染方法检测结肠组织中CD4+IL-9+T细胞比例;FCM检测脾脏、MLN、肠黏膜固有层分离的单个核细胞中CD4+IL-9+T细胞比例;ELISA方法检测血清中和脾脏、MLN、肠黏膜固有层分离的单个核细胞培养上清液中IL-9的分泌;Real time PCR检测IL-9 mRNA在结肠组织中的表达。4)Real time PCR检测慢性实验性结肠炎小鼠结肠组织中PU.1 mRNA的表达情况;Western blot检测慢性实验性结肠炎小鼠结肠组织中PU.1的蛋白表达。结果:1)给予IL-9抗体治疗后,与相应DSS/WT组和DSS/Tg组小鼠相比,IL-9 Ab/WT组和IL-9 Ab/Tg组小鼠体重明显回升,DAI评分和结肠大体形态学评分均明显下降,结肠长度更长;与DSS/Tg组小鼠相比,DSS/WT组小鼠缓解更明显。2)病理评分:给予IL-9抗体治疗后,IL-9Ab/WT组小鼠结肠炎细胞浸润明显减少,结构破坏减轻,黏膜得到修复,病理评分明显低于DSS/WT组小鼠(3.83±0.98 vs 11.00±0.63,P<0.05);与DSS/WT组小鼠相比,Isotype Ab/WT组小鼠结肠组织形态学评分(10.33±0.52 vs 11.00±0.63,P>0.05)无明显变化。同样,IL-9 Ab/Tg组小鼠结肠组织病理评分明显低于DSS/Tg组小鼠(6.33±0.82 vs 13.67±1.03,P<0.05);与DSS/Tg组小鼠相比,Isotype Ab/Tg组小鼠结肠组织病理评分(12.83±1.17 vs 13.67±1.03,P>0.05)无明显区别。同时,与IL-9 Ab/Tg组相比,IL-9 Ab/WT组小鼠病理评分更低(3.83±0.98 vs 6.33±0.82,P<0.05)。3)MPO活性:给予IL-9抗体治疗后,IL-9 Ab/WT组小鼠结肠组织MPO活性与DSS/WT组小鼠相比明显下降(0.13±0.02 vs 0.97±0.13,P<0.05);而Isotype Ab/WT组小鼠结肠组织MPO活性与DSS/WT组小鼠相比无明显变化(0.80±0.26 vs 0.97±0.13,P>0.05)。同样,IL-9 Ab/Tg组小鼠结肠组织MPO活性与DSS/Tg组小鼠相比明显下降(0.42±0.07 vs 1.94±0.12,P<0.05);而Isotype Ab/Tg组小鼠结肠组小鼠结肠组织MPO活性与DSS/Tg组小鼠相比无明显变化(1.89±0.36 vs 1.94±0.12,P>0.05)。同时,与IL-9Ab/Tg组相比,IL-9 Ab/WT组小鼠结肠组织MPO活性更低(0.13±0.02 vs0.42±0.07,P<0.05)。4)CD4和IL-9免疫荧光共染方法检测结肠组织中CD4+IL-9+T细胞的比例:给予IL-9抗体治疗后,IL-9 Ab/WT组小鼠结肠组织CD4+IL-9+T细胞的比例与DSS/WT组小鼠相比明显下降(0.20±0.04vs 0.32±0.03,P<0.05);而Isotype Ab/WT组小鼠结肠组织CD4+IL-9+T细胞的比例与DSS/WT组小鼠相比无明显变化(0.33±0.02 vs 0.32±0.03,P>0.05)。同样,IL-9 Ab/Tg组小鼠结肠组织CD4+IL-9+T细胞的比例与DSS/Tg组小鼠相比明显下降(0.29±0.02 vs 0.48±0.02,P<0.05);而Isotype Ab/Tg组小鼠结肠组小鼠结肠CD4+IL-9+T细胞的比例与DSS/Tg组小鼠相比无明显变化(0.44±0.04 vs 0.48±0.02,P>0.05)。同时,与IL-9 Ab/Tg组相比,IL-9 Ab/WT组小鼠结肠组织CD4+IL-9+T细胞的比例更低(0.20±0.04vs 0.29±0.02,P<0.05);FCM检测脾脏、MLN和肠黏膜固有层单个核细胞中CD4+IL-9+T细胞比例:给予IL-9抗体治疗后,IL-9 Ab/WT组小鼠脾脏单个核细胞中CD4+IL-9+T细胞比例与DSS/WT组小鼠相比明显下降(0.60%±0.03%vs 1.19%±0.12%,P<0.05);而Isotype Ab/WT组小鼠CD4+IL-9+T细胞比例与DSS/WT组小鼠相比无明显变化(1.21%±0.17%vs1.19%±0.12%,P>0.05);同样,IL-9 Ab/Tg组小鼠CD4+IL-9+T细胞比例与DSS/Tg组小鼠相比明显下降(0.87%±0.08%vs 2.26%±0.19%,P<0.05);而Isotype Ab/Tg组小鼠CD4+IL-9+T细胞比例与DSS/Tg组小鼠相比无明显变化(2.22%±0.24%vs 2.26%±0.19%,P>0.05);同时,与IL-9 Ab/Tg组相比,IL-9 Ab/WT组小鼠CD4+IL-9+T细胞比例更低(0.60%±0.03%vs0.87%±0.08%,P<0.05)。同样,在MLN和肠黏膜固有层单个核细胞中CD4+IL-9+T细胞比例与脾脏中单个核细胞中CD4+IL-9+T细胞比例变化一致。Real time PCR检测结肠组织中IL-9 mRNA的表达:给予IL-9抗体治疗后,IL-9 Ab/WT组小鼠结肠组织IL-9 mRNA表达与DSS/WT组小鼠相比明显下降(2.53±0.38 vs 3.56±0.78,P<0.05);而Isotype Ab/WT组小鼠结肠组织IL-9 mRNA表达与DSS/WT组小鼠相比无明显变化(3.60±0.74vs 3.56±0.78,P>0.05)。同样,IL-9 Ab/Tg组小鼠结肠组织IL-9 mRNA表达与DSS/Tg组小鼠相比明显下降(5.18±0.64 vs 6.32±0.74,P<0.05);而Isotype Ab/Tg组小鼠结肠组小鼠结肠IL-9 mRNA表达与DSS/Tg组小鼠相比无明显变化(6.35±0.68 vs 6.32±0.74,P>0.05)。同时,与IL-9 Ab/Tg组相比,IL-9 Ab/WT组小鼠结肠组织IL-9 mRNA表达更低(2.53±0.38 vs5.18±0.64,P<0.05)。ELISA方法检测血清中和脾脏、MLN、肠黏膜固有层分离的单个核细胞培养上清液中IL-9分泌水平:给予IL-9抗体治疗后,在血清中检测IL-9水平,可见IL-9 Ab/WT组IL-9分泌与DSS/WT组小鼠相比明显下降(119.12 pg/m L±6.90 pg/m L vs 261.05 pg/m L±20.30 pg/m L,P<0.05);而Isotype Ab/WT组IL-9分泌与DSS/WT组小鼠相比无明显变化(280.82 pg/m L±17.30 pg/m L vs 261.05 pg/m L±20.30 pg/m L,P>0.05);同样,IL-9 Ab/Tg组IL-9分泌与DSS/Tg组小鼠相比明显下降(278.59pg/m L±14.11 pg/m L vs 389.26 pg/m L±15.02 pg/m L,P<0.05);而Isotype Ab/Tg组IL-9分泌与DSS/Tg组相比无明显变化(381.92 pg/m L±12.34pg/m L vs 389.26 pg/m L±15.02 pg/m L,P>0.05)。同时,与IL-9 Ab/Tg组相比,IL-9 Ab/WT组IL-9分泌更低(119.12 pg/m L±6.90 pg/m L vs 278.59pg/m L±14.11 pg/m L,P<0.05)。在脾脏、MLN和肠黏膜固有层分离的单个核细胞培养上清液中检测IL-9分泌水平可得到一致结论。5)Real time PCR检测慢性实验性结肠炎小鼠结肠组织中PU.1 mRNA的表达:给予IL-9抗体治疗后,IL-9 Ab/WT组小鼠结肠组织PU.1 mRNA表达与DSS/WT组小鼠相比明显下降(1.98±0.34 vs 2.66±0.43,P<0.05);而Isotype Ab/WT组小鼠结肠组织PU.1 mRNA表达与DSS/WT组小鼠相比无明显变化(2.69±0.44 vs 2.66±0.43,P>0.05)。同样,IL-9 Ab/Tg组小鼠结肠组织PU.1 mRNA表达与DSS/Tg组小鼠相比明显下降(2.81±0.59 vs 4.75±0.86,P<0.05);而Isotype Ab/Tg组小鼠结肠组小鼠结肠PU.1 mRNA表达与DSS/Tg组小鼠相比无明显变化(4.80±0.86 vs 4.75±0.86,P>0.05)。同时,与IL-9 Ab/Tg组相比,IL-9 Ab/WT组小鼠结肠组织PU.1 mRNA表达更低(1.98±0.34 vs 2.81±0.59,P<0.05);Western blot检测慢性实验性结肠炎小鼠结肠组织中PU.1的蛋白表达:给予IL-9抗体治疗后,IL-9 Ab/WT组小鼠结肠组织PU.1蛋白表达与DSS/WT组小鼠相比明显下降(0.24±0.05 vs0.50±0.06,P<0.05);而Isotype Ab/WT组小鼠结肠组织PU.1蛋白表达与DSS/WT组小鼠相比无明显变化(0.51±0.06 vs 0.50±0.06,P>0.05)。同样,IL-9 Ab/Tg组小鼠结肠组织PU.1蛋白表达与DSS/Tg组小鼠相比明显下降(0.48±0.05 vs 0.71±0.07,P<0.05);而Isotype Ab/Tg组小鼠结肠组小鼠结肠PU.1蛋白表达与DSS/Tg组小鼠相比无明显变化(0.68±0.06 vs0.71±0.07,P>0.05)。同时,与IL-9 Ab/Tg组相比,IL-9 Ab/WT组小鼠结肠组织PU.1蛋白表达更低(0.24±0.05 vs 0.48±0.05,P<0.05)。结论:1在DSS诱导的慢性实验性结肠炎中Th9细胞分化及IL-9的表达明显增加,TL1A转基因组增加更为明显,提示TL1A在慢性实验性结肠炎发病过程中促进了Th9细胞的分化及IL-9的生成,进而促进慢性实验性结肠炎的发生。2 TL1A促进了Th9细胞的诱导分化;TL1A通过上调PU.1的表达,促进Th9细胞诱导分化及IL-9分泌,因此,TL1A调控PU.1的表达是其诱导Th9细胞分化及IL-9分泌的机制之一。3 IL-9抗体对慢性实验性结肠炎具有治疗作用,IL-9抗体在直接拮抗IL-9的同时,能够下调PU.1的表达,减少Th9细胞的分化,进一步导致IL-9生成减少。更多还原

【Abstract】 Inflammatory bowel disease(IBD)is a chronic gastrointestinal inflammatory condition consisting of Crohn’s disease(CD)and ulcerative colitis(UC).Environmental,genetic and microbial factors with the immune system result in dysregulated immune responses in IBD.However,the mechanism still remains unclear and the treatment is far from effective.Further research in pathogenesis of IBD should be conducted to provide new theraputic targets.Intestine mucosal dysimmunity plays a core role in the pathogenesis of IBD.CD4+T cells are the key effector cells in immune system.CD4+T cells are composed of six main subgroups: Th1 cells,Th2 cells,Th17 cells,Th22 cells,newly discovered Th9 cells,and Treg cells.Each group of cells secretes specific cytokines.PU.1,an ETS family transcription factor,also identified as the spleen focus forming virus proviral integration site-1(Sfpi1)is a key transcription factor in the process of Th9 cell activation.The CD4 Na?ve T cells simulated with IL-4,TGF-β and IL-2 differentiate into Th9 cells in vitro.Th9 cells can deteriorate a variety of autoimmune inflammation via secreting specific pro-inflammatory cytokine IL-9.Some clinical researches have shown that Th9 cells and IL-9 increased significantly in IBD patients.More importantly,the expression of IL9 mRNA and IL-9 protein in patients with active UC was higher than in control subject.It has been demonstrated that Th9 cells and IL-9 play a key role in IBD.Currently,TL1 A,a tumor necrosis factor superfamily member,has been identified as one of IBD susceptibility genes.TL1 A,lymphocyte costimulator,can induce T cells proliferation and activation.TL1 A plays a critical role in various diseases such as asthma,rheumatoid arthritis and IBD.Papadakis found that TL1 A synergizes with IL-12 and IL-18 enhanced IFN-γ production in human T cells and NK cells.TL1A was found to enhance the Th1,Th2 and Th17 cells responses in chronic colitis model.However,the association between Th9 cells and IBD is still unknown.lymphocytes over-expressed TL1 A transgenic mice and WT mice were used in this study,DSS-induced chronic murine colitis model was established and splenic cells and mesenteric lymph node(MLN)were isolated,purified and differentiated into Th9 cells and treated by IL-9 antibody and TL1 A antibody after TL1 A stimulation for investigation of the relationship between TL1 A and Th9,which might provide a new evidence for prevention and treatment of IBD.The experiment mainly consists of the following three parts: Part one: The changes of Th9 cells and IL-9 expression in chronic experimental colitis of sustained TL1 A expressionObjective: To study the effects of Th9 cells and IL-9 on chronic experimental colitis of sustained TL1 A expression.Methods: 1)LCK-CD2-TL1A-GFP-transgenic mice were identified by real-time PCR.2)The WT and Tg mice were randomly divided as followed:(1)Control/WT group,(2)DSS/WT group,(3)Control/Tg group,(4)DSS/Tg group.Chronic colitis was induced by drinking 2.5% DSS from 1 to 5 days,8 to 12 days,15 to 19 days and 22 to 26 days,and distilled water was given during the remaining time.Animals were sacrificed at 29 th day.3)Severity of colitis was evaluated by body weight(BW)changes,disease activity index(DAI),colon length,colon histology changes and pathology score.4)The percentage of CD4+IL-9+ T cells in colon was detected by immunefluorescence staining.The percentages of CD4+IL-9+ T cells in spleen,mesenteric lymph node(MLN)and lamina propria mononuclear cells were detected by Flow cytometry instrument(FCM).The expression of IL-9 mRNA was detected by Real-time PCR.The secretion level of IL-9 of serum,spleen,mesenteric lymph node(MLN)and lamina propria mononuclear cells(LPMCs)was detected by ELISA.5)The expression of TGF-β and IL-4 mRNA in colon was detected by Real-time PCR.The secretion level of TGF-β and IL-4 of spleen,MLN and lamina propria mononuclear cells mononuclear cells was detected by ELISA.6)The protein expression of TL1 A and PU.1 was detected by Western blot assay.The expression of TL1 A and PU.1 mRNA was detected by Real-time PCR.Results: 1)The TLlA DNA located at 192 bp in Tg mice and did not express at 192 bp in WT mice.2)Significantly increased DAI,reduced BW and colon length were observed in DSS/WT group and DSS/Tg group as compared to corresponding Control/WT group and Control/Tg group.More severe lesions in DSS/Tg as compared to DSS/WT group.3)Worsened inflammation characterized by increased cellular infiltrate,mucin depletion,crypt abscess,and architectural changes in DSS/WT group and DSS/Tg group as compared to corresponding Control/WT group and Control/Tg group(8.25±0.50 vs 0.00±0.00,P<0.05)and(12.50±0.58 vs 0.00±0.00,P<0.05).Significantly increased histological score was observed in DSS/Tg group as compared to DSS/WT group(12.50±0.58 vs 8.25±0.50,P<0.05).4)MPO activity: Significantly increased MPO activity was observed in DSS/WT group as compared to Control/WT group(1.09±0.21 vs 0.43±0.11,P<0.05).Significantly increased MPO activity was observed in DSS/Tg group as compared to Control/Tg group(1.80±0.12 vs 0.60±0.07,P <0.05).Significantly increased MPO activity was observed in DSS/Tg group as compared to DSS/WT group(1.80±0.12 vs 1.09±0.21,P <0.05).5)The percentage of CD4+IL-9+ T cells was detected by immunofluorescence staining.Significantly increased CD4+IL-9+ T cells were observed in DSS/WT group as compared to Control/WT group(0.24±0.03 vs 0.09±0.01,P<0.05).The expression of CD4+IL-9+ T cells in DSS/Tg group mice were higher than that in Control/Tg(0.44±0.03 vs 0.10±0.01,P<0.05).Significantly increased CD4+IL-9+ T cells was observed in DSS/Tg group as compared to DSS/WT group(0.44±0.03 vs 0.24±0.03,P<0.05).The results of between the percentages of CD4+IL-9+ T cells in spleen,mesenteric lymph node(MLN)and lamina propria mononuclear cells were detected by FCM,the secretion level of IL-9 of serum,spleen,mesenteric lymph node(MLN)and lamina propria mononuclear cells(LPMCs)were detected by ELISA,the expression of IL-9 mRNA in colon was detected by Real-time PCR and the result of immunofluorescence staining are consistent.6)The expression of TGF-β and IL-4 mRNA was detected by Real-time PCR.Significantly increased expression of TGF-β and IL-4 mRNA was observed in DSS/WT group(6.22±0.53,3.35±0.57)as compared to corresponding Control/WT group(1.02±0.20,1.01±0.17)(P<0.05).The expression of TGF-β and IL-4 mRNA in DSS/Tg group(15.06±1.63,4.80±0.89)was higher than that in Control/WT group(1.16±0.24,1.29±0.33)(P<0.05).Significantly increased expression of TGF-β and IL-4 mRNA was observed in DSS/Tg group(15.06±1.63,4.80±0.89)as compared to DSS/WT group(6.22±0.53,3.35±0.57)(P<0.05).The results of between the level of TGF-β and IL-4 of spleen,MLN and lamina propria mononuclear cells was detected by ELISA and the expression of TGF-β and IL-4 mRNA was detected by Real-time PCR are consistent.7)The expression of TL1 A and PU.1 mRNA was detected by Real-time PCR.Significantly increased expression of TL1 A and PU.1 mRNA was observed in DSS/WT group(9.19±1.24,2.68±0.46)as compared to corresponding Control/WT group(1.01±0.14,1.03±0.26)(P<0.05).The expression of TL1 A and PU.1 mRNA in DSS/Tg group(12.62±2.01,4.90±0.87)was higher than that in Control/WT group(1.21±0.16,1.20±0.18)(P<0.05).Significantly increased expression of TL1 A and PU.1 mRNA was observed in DSS/Tg group(12.62±2.01,4.90±0.87)as compared to DSS/WT group(9.19±1.24,2.68±0.46)(P<0.05).The results of between the expression of TL1 A and PU.1 protein in colon were detected by ELISA and the expression of TL1 A and PU.1 mRNA was detected by Real-time PCR are consistent.Part two: The role and mechanism of TL1 A in the differentiation of Th9 cellObjective: To research the differentiation of Th9 cell by TL1 A.Methods: 1)CD4 Na?ve T cells were isolated by magnetic cell sorting(MACS)negative sorting from spleen and mesenteric lymph node(MLN)mononuclear cells.The percentage of CD3~+CD4~+CD44-CD62L+ T cells(CD4 Na?ve T cells)was detected by FCM.2)CD4 Na?ve T cells were isolated from spleen and MLN mononuclear cells.Subsequently,CD4 Na?ve T cells were stimulated to Th9 cells.The percentage of CD4+IL-9+ T cells of before sorting cells and Th9 cells was detected by FCM.3)After stimulation of TL1 A with 100 ng/mL,the TL1 A antibody with 4 ug/mL or IL-9 antibody with 50 ng/mLwere further administrated in the process of Th9 cells differentiation.The percentage of CD4+IL-9+ T cells of Th9 cells from spleen and MLN mononuclear cells was detected by FCM.The level of CD4+IL-9+ T cells of Th9 cells from spleen and MLN mononuclear cells was detected by ELISA.The expression of PU.1 of Th9 cells from spleen and MLN mononuclear cells was detected by Western blot assay.Results: 1)The percentages of CD3~+CD4~+CD44-CD62L+ T cells(CD4 Na?ve T cells)were detected from spleen and MLN by FCM.The purity of the sorted cells from spleen and MLN indicated the isolated cells could used in the following experiment(90.18%±2.62%)and(92.12%±2.82%).2)The percentage of CD4+IL-9+ T cells of before sorting cells and Th9 cells from spleen was detected by FCM.Significantly increased percentage of IL-9 was observed in the both Th9 cells/WT and Th9 cells/Tg group(20.93%±3.60%,31.40%±3.66%)as compared to the corresponding before sorting cells/WT and before sorting cells/Tg group(0.28%±0.10%,0.42%±0.10%)(P<0.05).Meanwhile,significantly increased percentage of CD4+IL-9+ T cells was observed in the Th9 cells/Tg as compared to the Th9 cells/WT(31.40%±3.66% vs 20.93%±3.60%,P<0.05).The percentage of CD4+IL-9+ T cells from MLN and spleen before and after sorting was detected by FCM.3)After administration of TL1 A with 100 ng/mL,the percentage of CD4+IL-9+ T cells and secretion level of IL-9 and protein expression of PU.1 from spleen and MLN mononuclear cells were detected.The Th9 cells from spleen mononuclear cells: The percentage of CD4+IL-9+ T cells and secretion level of IL-9 and protein expression of PU.1 of the TL1A/WT group(CD4+IL-9+ T cells: 30.63%±2.32% vs 21.07%±2.35%,P<0.05;IL-9: 3867.63 pg/mL±88.11 pg/mL vs 2464.15 pg/mL±122.63 pg/mL,P<0.05;PU.1: 1.25±0.08 vs 0.95±0.06,P<0.05)and TL1A/Tg group(CD4+IL-9+ cells: 37.63%±2.26% vs 29.90%±2.33%,P<0.05;IL-9: 4221.90 pg/mL±81.11 pg/mL vs 2855.90 pg/mL±128.59 pg/mL,P<0.05;PU.1: 1.42±0.07 vs 1.18±0.06,P<0.05)were significantly increased as compared to the corresponding control/WT and control/Tg group.Meanwhile,further increase were observed in the TL1A/Tg groups as compared to the TL1A/WT groups(P<0.05).The Th9 cells from MLN mononuclear cells: The percentage of CD4+IL-9+ T cells and secretion level of IL-9 and protein expression of PU.1 of the TL1A/WT group(CD4+IL-9+ T cells: 29.80%±2.3% vs 24.60%±1.67%,P<0.05;IL-9: 3939.17 pg/mL±166.15 pg/mL vs 2603.82 pg/mL±98.20 pg/mL,P<0.05;PU.1: 1.23±0.06 vs 0.93±0.06,P<0.05)and TL1A/Tg group(CD4+IL-9+ T cells: 42.67%±2.56% vs 31.40%±2.75%,P<0.05;IL-9: 4337.73 pg/mL±161.69 pg/mL vs 3142.04 pg/mL±153.75 pg/mL,P<0.05;PU.1: 1.41±0.08 vs 1.22±0.08,P<0.05)were significantly increased as compared to the corresponding control/WT group and control/Tg group.Meanwhile,further increase were observed in the TL1A/Tg groups as compared to the TL1A/WT groups(P<0.05).4)After administration of TL1 A antibody with 4 ug/mL,the percentage of CD4+IL-9+ T cells and secretion level of IL-9 and protein expression of PU.1 from spleen and MLN mononuclear cells were detected.The Th9 cells from spleen mononuclear cells: The percentage of CD4+IL-9+ T cells and secretion level of IL-9 and protein expression of PU.1 of the TL1 A Ab/WT group(CD4+IL-9+ T cells: 18.30%±1.51% vs 30.63%±2.32%,P<0.05;IL-9: 2556.13 pg/mL±83.65 pg/mL;PU.1: 0.63±0.07 vs 1.25±0.08,P<0.05)and TL1 A Ab/Tg group(CD4+IL-9+ T cells: 23.67%±2.06% vs 37.63%±2.26%,P<0.05;IL-9: 2556.13 pg/mL±83.65 pg/mL vs 3867.63 pg/mL±88.11 pg/mL,P<0.05;PU.1: 0.85±0.07 vs 1.42±0.07,P<0.05)were significantlyly reduced as compared to the corresponding TL1A/WT and TL1A/Tg group.Meanwhile,further decrease were observed in the TL1 A Ab/WT groups as compared to the TL1 A Ab/Tg groups(P<0.05).The Th9 cells from MLN mononuclear cells: The percentage of CD4+IL-9+ T cells and secretion level of IL-9 and protein expression of PU.1 of the TL1 A Ab/WT group(CD4+IL-9+ T cells: 17.33%±1.96% vs 29.80%±2.30%,P<0.05;IL-9: 2576.57 pg/mL±53.10 pg/mL vs 3939.17 pg/mL±166.15 pg/mL,P<0.05;PU.1: 0.63±0.09 vs 1.23±0.06,P<0.05)and TL1 A Ab/Tg group(CD4+IL-9+ T cells: 29.50%±2.01% vs 42.27%±2.56%,P<0.05;IL-9: 3111.39 pg/mL±62.44 pg/mL vs 4337.73 pg/mL±161.69 pg/mL,P<0.05;PU.1: 0.92±0.10 vs 1.41±0.08,P<0.05)were significantly decreased as compared to the corresponding TL1A/WT and TL1A/Tg group.Meanwhile,further decrease were observed in the TL1 A Ab/WT groups as compared to the TL1 A Ab/Tg groups(P<0.05).5)After administration of IL-9 antibody with 50 ng/mL,the percentage of CD4+IL-9+ T cells and secretion level of IL-9 and protein expression of PU.1 from spleen and MLN mononuclear cells were detected.The Th9 cells from spleen mononuclear cells: The percentage of CD4+IL-9+ T cells and secretion level of IL-9 and protein expression of PU.1 of the IL-9 Ab/WT group(CD4+IL-9+ T cells: 23.50%±1.93% vs 30.63%±2.32%,P<0.05;IL-9: 2218.88 pg/mL±90.83 pg/mL vs 3867.63 pg/mL±88.11 pg/mL,P<0.05;PU.1: 0.76±0.08 vs 1.25±0.08,P<0.05)and IL-9 Ab/Tg group(CD4+IL-9+ T cells: 26.77%±1.90% vs 37.63%±2.26%,P<0.05;IL-9: 2669.20 pg/mL±100.65 pg/mL vs 4221.90 pg/mL±81.11 pg/mL,P<0.05;PU.1: 0.94±0.06 vs 1.42±0.07,P<0.05)were significantly reduced as compared to the corresponding TL1A/WT and TL1A/Tg group.Meanwhile,further decrease were observed in the IL-9 A b/WT groups as compared to the IL-9 Ab/Tg groups(P<0.05).The Th9 cells from MLN mononuclear cells: The percentage of CD4+IL-9+ T cells and secretion level of IL-9 and protein expression of PU.1 of the IL-9 Ab/WT group(CD4+IL-9+ T cells: 24.70%±2.10% vs 29.80%±2.30%,P<0.05;IL-9: 2215.48 pg/mL±129.40 pg/mL vs 3939.17 pg/mL±166.15 pg/mL,P<0.05;PU.1: 0.66±0.07 vs 1.23±0.06,P<0.05)and IL-9 Ab/Tg group(CD4+IL-9+ T cells: 30.20%±2.40% vs 42.27%±2.56%,P<0.05;IL-9: 2719.64 pg/mL±61.32 pg/mL vs 4337.73 pg/mL±161.69 pg/mL,P<0.05;PU.1: 0.88±0.08 vs 1.41±0.08,P<0.05)were significantly decreased as compared to the corresponding TL1A/WT and TL1A/Tg group.Meanwhile,further decrease were observed in the IL-9 Ab/WT groups as compared to the IL-9 Ab/Tg groups(P<0.05).Part three: The effect of IL-9 antibody on chronic experimental colitis of sustained TL1 A expression.Objective: To explore the effects of IL-9 antibody on chronic experimental colitis of sustained TL1 A expression.Methods: 1)The WT and Tg mice were randomly divided as followed:(1)Control/WT group,(2)DSS/WT group,(3)Ab Isotype/WT group,(4)IL-9 Ab/WT group,(5)Control/Tg group,(6)DSS/Tg group,(7)Ab Isotype/Tg group,(8)IL-9 Ab/Tg group.Chronic colitis was induced by drinking 2.5% DSS from 1 to 5 days,8 to 12 days,15 to 19 days and 22 to 26 days,and distilled water was given during the remaining time.In IL-9 Ab group and IL-9 Ab Isotype group,100 ug IL-9 antibody or control immunoglobulin G was injected into mice intraperitoneally every other day from the day 14 through day 29.Animals were sacrificed at 29 th day.2)Severity of colitis was evaluated by body weight(BW)changes,disease activity index(DAI),colon length,colon histology changes and pathology score.3)The percentage of CD4+IL-9+ T cells in colon was detected by immunofluorescence staining.The percentages of CD4+IL-9+ T cells in spleen,mesenteric lymph node(MLN)and lamina propria mononuclear cells were detected by Flow cytometry instrument(FCM).The expression of IL-9 mRNA was detected by Real-time PCR.The secretion level of IL-9 of serum,spleen,MLN and LPMCs was detected by ELISA.4)The expression of PU.1 was detected by Western blot assay.The expression of PU.1 mRNA was detected by Real-time PCR.Results: 1)Body weight(BW)change and disease activity index(DAI).After administration of IL-9 antibody,significantly increased BW and reduced DAI were observed in the both IL-9 Ab/WT group and IL-9 Ab/Tg group as compared to the corresponding DSS/WT group and DSS/Tg group.Meanwhile,significantly increased BW and reduced DAI were observed in the IL-9 Ab/Tg group as compared to the IL-9 Ab/WT group.There was no differerce in BW and DAI between Ab Isotype/WT group and DSS/WT group or Ab Isotype/Tg group and DSS/Tg group(P>0.05).2)H﹠E staining(×100)and histological score.After administration of IL-9 antibody,the lymphocyte cellular infiltrate and mucosal structure damage were alleviated.Significantly reduced histological score were observed in the both IL-9 Ab/WT group and IL-9 Ab/Tg group(3.83±0.98,6.33±0.82)as compared to the corresponding DSS/WT group and DSS/Tg group(11.00±0.63,13.67±1.03)(P<0.05).Meanwhile,significantly reduced histological score were observed in the IL-9 Ab/WT group as compared to the IL-9 Ab/Tg group(3.83±0.98 vs 6.33±0.82,P<0.05).There was no differerce in histological score between Ab Isotype/WT group and DSS/WT group(10.33±0.52 vs 11.00±0.63,P>0.05)or Ab Isotype/Tg group and DSS/Tg group(12.83±1.17 vs 13.67±1.03,P>0.05).3)MPO activity: After administration of IL-9 antibody.Significantly reduced MPO activity were observed in the both IL-9 Ab/WT group and IL-9 Ab/Tg group(0.13±0.02,0.42±0.07)as compared to the corresponding DSS/WT group and DSS/Tg group(0.97±0.13,1.94±0.12)(P<0.05).Meanwhile,significantly reduced MPO activity were observed in the IL-9 Ab/WT group as compared to the IL-9 Ab/Tg group(0.13±0.02 vs 0.42±0.07,P<0.05).There was no differerce in MPO activity between Ab Isotype/WT group and DSS/WT group(0.80±0.26 vs 0.97±0.13,P>0.05)or Ab Isotype/Tg group and DSS/Tg group(1.89±0.36 vs 1.94±0.12,P>0.05).4)The percentage of CD4+IL-9+ T cells in colon was detected by immunofluorescence staining.After administration of IL-9 antibody.Significantly reduced CD4+IL-9+ T cells were observed in the both IL-9 Ab/WT group and IL-9 Ab/Tg group(0.20±0.04,0.29±0.02)as compared to the corresponding DSS/WT group and DSS/Tg group(0.48±0.02,0.32±0.03)(P<0.05).Meanwhile,significantly reduced CD4+IL-9+ T cells were observed in the IL-9 Ab/WT group as compared to the IL-9 Ab/Tg group(0.20±0.04 vs 0.29±0.02,P<0.05).There was no differerce in CD4+IL-9+ T cells between Ab Isotype/WT group and DSS/WT group(0.33±0.02 vs 0.32±0.03,P>0.05)or Ab Isotype/Tg group and DSS/Tg group(0.44±0.04 vs 0.48±0.02,P>0.05).The percentages of CD4+IL-9+ T cells in spleen,mesenteric lymph node(MLN)and lamina propria mononuclear cells were detected by FCM,the level of IL-9 of serum,spleen,MLN and LPMCs was detected by ELISA,the expression of IL-9 mRNA in colon was detected by Real-time PCR and the result of immunofluorescence staining are consistent.5)The expression of PU.1 mRNA was detected by Real-time PCR.After administration of IL-9 antibody,significantly reduced expression of PU.1 mRNA were observed in the both IL-9 Ab/WT group and IL-9 Ab/Tg group(1.98±0.34,2.81±0.59)as compared to the corresponding DSS/WT group and DSS/Tg group(2.66±0.43,4.75±0.86)(P<0.05).Meanwhile,significantly reduced expression of PU.1 mRNA were observed in the IL-9 Ab/WT group as compared to the IL-9 Ab/Tg group(1.98 ±0.34 vs 2.81±0.59,P<0.05).There was no differerce in expression of PU.1 mRNA between Ab Isotype/WT group and DSS/WT group(2.69±0.44 vs 2.66±0.43,P>0.05)or Ab Isotype/Tg group and DSS/Tg group(4.80±0.86 vs 4.75±0.86,P>0.05).The results of between the expression of PU.1 protein in colon were detected by ELISA,the expression of PU.1 mRNA was detected by Real-time PCR and the expression of TL1 A mRNA was detected by Real-time PCR are consistent.Conclusions:1 Significantly increased of Th9 cells number and IL-9 production was observed in chronic experimental colitis of sustained TL1 A expression.TL1 A induced Th9 cells differentiation and IL-9 production in chronic experimental colitis.2 TL1 A induced Th9 cells differentiation and IL-9 production by upregulating PU.1 expression.On the contrary,TL1 A antibody inhibited Th9 cells differentiation and decreased IL-9 production by downregulating the expression of PU.1.3 IL-9 antibody significantly alleviated colonic inflammation by suppressing PU.1 expression and reducing Th9 cells differentiation and IL-9 production.更多还原