雷公藤多苷上调克罗恩病病人局部肠粘膜Treg细胞及调节炎症因子的分泌

发布时间：2018-05-30 02:22

本文选题：克罗恩病 + 雷公藤多苷　；参考：《南京大学》2014年硕士论文

【摘要】：背景：克罗恩病(Crohn's disease, CD)属于炎症性肠病(inflammatory bowel disease,IBD)的一种,近一个世纪以来其发病率越来越高。尽管相关研究逐渐深入,但其确切病因至今仍然不清。目前认为其病因涉及基因易感性、环境因素、免疫因素。表达叉状头／翅膀状螺旋转录因子(Forkhead Box P3, Foxp3)的调节性T细胞(t regulatory cells, Tregs)通过分泌IL-10、TGF-β发挥抑炎作用,在维持机体免疫稳态方面发挥重要作用。许多研究发现Tregs与CD发病之间存在相关性。雷公藤多苷提取自中国一味传统中药雷公藤的根部,具有抗炎、免疫调节、抗肿瘤等作用。在中国,雷公藤多苷被广泛用于治疗多种自身免疫性疾病,包括类风湿关节炎、肾炎及系统性红斑狼疮等。雷公藤能够缓解结肠炎小鼠的肠道炎症,其作用机制包括抑制TLRs/NF-κB和TNF-α/TNFR2信号通路,抑制L-6/STAT3通路、下调IL-17表达。我们之前研究证实,雷公藤多苷能够有效诱导CD缓解。但其具体机制尚未明确。目的：探索雷公藤多苷对CD病人肠道粘膜局部炎症因子表达及Foxp3+ Tregs水平的作用。方法：从南京军区南京总医院门诊部入选处于中重度活动期的CD病人,给予口服雷公藤多苷片60mg／天(中国江苏,泰州制药厂)。入选时及三个月后行结肠镜检查,于病变明显的同一段肠管钳取6块肠粘膜。HE染色后使用光镜观察肠粘膜病理变化并进行炎症浸润程度的评分；采用免疫组化法检测病变肠粘膜中Foxp3、IL-10、TNF-α的表达变化；采用免疫印迹(Western blot)检测Foxp3蛋白表达的变化；采用酶联免疫吸附法(ELISA)测定病变肠道粘膜中炎症因子IL-10、TNF-α水平变化。结果： 20个CD病人参与了本项研究,年龄介于25-57岁之间。其中8个病人疾病处于轻度活动期,另外12个病人疾病处于中度活动期。所有人均接受了三个月的口服雷公藤多苷片治疗,没有人因为疾病不良反应而退出实验。治疗前的病变肠粘膜HE病理可见溃疡、上皮损害、腺体减少和炎症细胞的浸润,而接受三个月雷公藤多苷片治疗后,镜下病理明显改善。治疗后的炎症浸润程度评分显著降低(8.1±2.2 vs 6.8±2.6,P0.05)。免疫组化的结果表明,雷公藤治疗后,肠粘膜中Foxp3+ Tregs和IL-10阳性细胞的水平呈现增多趋势,而促炎因子TNF-α则呈现下调趋势。Western blot检测显示,治疗前病变肠粘膜中Foxp3蛋白有一定的表达,而雷公藤多苷治疗后,Foxp3蛋白能够进一步上调。在炎症因子表达方面,雷公藤多苷使促炎因子TNF-α水平显著下降(189.6±17.4 vs173.0±19.0,P0.01),而抑炎因子IL-10的水平显著上升(45.7±16.3 vs 55.6±14.8,P0.05)。结论：雷公藤多苷能够缓解CD病变肠管的炎症,改善局部粘膜的病理损害,调节局部免疫环境的炎症因子的表达水平,使促炎因子TNF-1α水平下降,抑炎因子IL-10的水平上升,其作用机制可能和病变肠管中Foxp3+ Tregs增多有关。
[Abstract]:Background: Crohn's disease (CDD) is a kind of inflammatory bowel disease. The exact cause of the disease is still unclear, although the relevant studies have gradually deepened. It is believed that the etiology is related to genetic susceptibility, environmental factors and immune factors. Regulatory T cell regulatory cells, Tregs), which expresses Forkhead Box P3 (Foxp3), plays an important role in maintaining immune homeostasis by secreting IL-10 TGF- 尾. Many studies have found a correlation between Tregs and CD. Tripterygium wilfordii polyglycosides extracted from the roots of traditional Chinese medicine Tripterygium wilfordii have anti-inflammatory, immunomodulatory and anti-tumor effects. In China, tripterygium wilfordii polyglycosides are widely used in the treatment of many autoimmune diseases, including rheumatoid arthritis, nephritis and systemic lupus erythematosus. Tripterygium wilfordii can relieve intestinal inflammation in mice with colitis by inhibiting TLRs / NF- 魏 B and TNF- 伪 / TNFR2 signaling pathway, inhibiting L-6/STAT3 pathway and down-regulating IL-17 expression. Our previous studies have confirmed that triptolide can effectively induce CD remission. But its specific mechanism is not clear. Objective: to investigate the effect of tripterygium wilfordii polyglycoside on the expression of local inflammatory factors and the level of Foxp3 Tregs in intestinal mucosa of CD patients. Methods: CD patients were selected from the outpatient department of Nanjing General Hospital of Nanjing military region and given oral Tripterygium wilfordii polyglycoside tablets for 60mg/ days (Taizhou Pharmaceutical Factory, Jiangsu, China). Colonoscopy was performed at the time of selection and after 3 months. After 6 intestinal mucosa was stained with HE, the pathological changes of intestinal mucosa were observed with light microscope and the degree of inflammatory infiltration was evaluated. The expression of Foxp3hIL-10 TNF- 伪 was detected by immunohistochemistry, the expression of Foxp3 protein was detected by Western blot, and the level of IL-10TNF- 伪 in intestinal mucosa was detected by enzyme-linked immunosorbent assay (Elisa). Results: twenty CD patients, aged 25-57, participated in the study. Among them, 8 patients had mild active disease and 12 patients had moderate active disease. All were treated with oral tripterygium wilfordii for three months, and no one dropped out because of adverse effects. The pathological changes of intestinal mucosa before treatment were ulcer, epithelial damage, decreased glands and infiltration of inflammatory cells. After three months of treatment with tripterygium wilfordii polyglycosides, the pathological changes were obviously improved under microscope. After treatment, the score of inflammatory infiltration decreased significantly (8.1 卤2.2 vs 6.8 卤2.6 P 0.05). The results of immunohistochemistry showed that the levels of Foxp3 Tregs and IL-10 positive cells in intestinal mucosa increased after Tripterygium wilfordii treatment, while the levels of TNF- 伪 were down-regulated. Western blot showed that TNF- 伪 was down-regulated. Foxp3 protein was expressed in intestinal mucosa before treatment, but Foxp3 protein was upregulated further after triptolide was treated with tripterygium wilfordii. In terms of inflammatory factor expression, tripterygium wilfordii polyglycoside significantly decreased the level of proinflammatory factor TNF- 伪 by 189.6 卤17.4 vs173.0 卤19.0U P0.01g, while the level of IL-10 increased significantly by 45.7 卤16.3 vs 55.6 卤14.8P0.05a. Conclusion: Tripterygium wilfordii polyglycoside can alleviate the inflammation of intestinal canal of CD lesion, improve the pathological damage of local mucosa, regulate the expression of inflammatory factor in local immune environment, decrease the level of proinflammatory factor TNF-1 伪 and increase the level of IL-10. The mechanism may be related to the increase of Foxp3 Tregs in the diseased intestine.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R574.62

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