细胞间粘附分子-1修饰的间充质干细胞对于炎症性肠病的治疗作用

发布时间：2018-03-01 07:37

本文关键词： 炎症性肠病间充质干细胞细胞间粘附分子-1 细胞治疗靶向作用　出处：《河北北方学院》2016年硕士论文　论文类型：学位论文

【摘要】：间充质干细胞(Mesenchymal stem cells,MSCs)移植治疗炎症性肠病(inflammatory bowel diseases,IBD)已经成为IBD治疗领域有发展前途的治疗策略。目前主流研究认为IBD的发病机制主要与非致病性肠抗原作用于遗传易感者引起的免疫应答失调有关,最终引起肠道黏膜屏障功能障碍进而导致肠组织损伤。MSCs具有免疫调节和促组织修复的生物学特性,基于此,MSCs已经被尝试应用于治疗免疫相关疾病,并且取得了良好的疗效。目前研究表明MSCs能够归巢至受损肠道组织,有效地促进黏膜修复,缓解IBD的临床症状。此外,MSCs治疗IBD具有良好的安全性。因此,MSCs疗法为临床治疗IBD,尤其是严重难治IBD提供了一个较好的选择。但是,目前MSCs的临床应用还存在着治疗效率不高,疗效不够稳定等不足,因此未来的研究应着眼于优化MSCs的治疗效果,提高MSCs移植治疗的稳定性等。MSCs的免疫抑制功能主要通过分泌可溶性分子和表达细胞表面粘附分子来实现。更重要的是,MSCs的定向迁移能力取决于配体和受体间的相互作用。细胞间粘附分子-1(Intercellular adhesion molecules 1,ICAM-1)是MSCs发挥功能的关键分子,生理状态下MSCs表达较低水平的ICAM-1,当处于炎性微环境中时,MSCs显著上调其细胞表面的ICAM-1,进一步研究表明粘附分子基因被敲除或封闭功能后,MSCs的免疫抑制作用显著降低。我们在前期工作中通过细胞转染技术提高MSCs表面ICAM-1表达量,构建了稳定高表达ICAM-1的MSCs细胞株。相关研究结果显示ICAM-1修饰的MSCs可在体外发挥强大免疫抑制功能。但是,ICAM-1修饰MSCs能否在体内发挥免疫调节能力,其是否能够优化IBD的治疗效果目前尚未见报道。基于此,我们建立了稳定的动物模型,考察ICAM-1修饰的MSCs对于IBD的体内疗效,并进一步探索其内在机制。我们建立了经典的葡聚糖硫酸钠(dextran sulfate sodium,DSS)诱导的IBD小鼠模型。通过自由饮用不同浓度的DSS溶液,探讨不同浓度的DSS溶液对于炎症性肠病小鼠模型的影响;鼠尾静脉输注ICAM-1修饰的MSCs对IBD小鼠进行治疗,通过体内外实验探讨其对IBD的治疗作用。(1)雄性BALB/c小鼠随机分为对照组和3%-DSS,5%DSS,7%-DSS饮用组。观察各项指标,系统探讨不同浓度DSS诱导的IBD小鼠模型,以期建立可重复、稳定高、可操作性优良的模型,从而为IBD的细胞治疗打下坚定的基础。(2)体外培养原代MSCs及C3细胞系,应用流式细胞术检测C3细胞系ICAM-1的表达量,并体外扩增及冻存细胞,以备后续实验应用。(3)细胞治疗,实验分组:Control组,PBS组,MSCs组,C3组,C3-MIGRI组,C3-ICAM-1组,建模第3天进行细胞鼠尾静脉注射移植,注射量为500μl浓度为5×106个/ml的细胞混悬液,冰冻切片观察MSCs在小鼠体内分布,观察各组小鼠一般情况、生存率,检测靶器官病理变化,IBD相关免疫因子表达水平和Th细胞亚群比例。饮用3%,5%,7%浓度DSS的小鼠在第6天结肠均有不同程度溃疡形成,成模率随着DSS浓度提升而增加,但是小鼠死亡率也相应增加,饮用5%浓度的DSS溶液有助于高效、经济地建立IBD小鼠模型,为MSCs治疗IBD打下了坚定的基础。细胞归巢实验显示过表达ICAM-1可显著提高MSCs靶向归巢效率,进而发挥组织修复和免疫调节功能,ICAM-1修饰的MSCs可促进IBD小鼠精神、饮食、体重和活动等一般状况恢复,提高生存率,减轻病理损伤,影响Th细胞亚群的分化,降低脾脏中IFN-γ,IL-17的水平,增加Foxp3的水平。综上所述,本研究成功的建立了DSS诱导的IBD小鼠模型,ICAM-1修饰的MSCs可通过促靶向作用以及免疫抑制作用,有效地增强了MSCs对于IBD的治疗效果,拓宽了IBD细胞治疗的领域。
[Abstract]:Mesenchymal stem cells (Mesenchymal stem cells, MSCs) transplantation for the treatment of inflammatory bowel disease (inflammatory bowel, diseases, IBD) has become the treatment strategy for IBD field promising. The current mainstream studies suggest that immune response in the pathogenesis of IBD and non pathogenic intestinal antigen on genetic susceptibility caused by disorders eventually lead to intestinal mucosal barrier function leading to.MSCs intestinal tissue injury with immune regulation and biological characteristics, promote tissue repair on the basis of this, MSCs has been used to treat immune related diseases, and achieved good results. The present study shows that MSCs can migrate into the damaged intestinal mucosa, effectively promote mucosal repair, clinical remission the symptoms of IBD. In addition, MSCs treatment of IBD has good security. Therefore, MSCs therapy for the treatment of IBD, especially the severe refractory IBD provides a Good choice. However, the clinical application of MSCs still exist in the treatment efficiency is not high, the effect is not stable enough, so the future research should focus on the optimization of MSCs treatment effect, improve the stability of MSCs transplantation for the treatment of immune suppression of.MSCs function by secreting soluble molecules and expression of cell surface adhesion molecules to achieve. More importantly, the interaction of MSCs depends on the directional migration of ligands and receptors. Intercellular adhesion molecule -1 (Intercellular adhesion molecules 1, ICAM-1) is a key molecule in MSCs function, physiological state of MSCs expressed low levels of ICAM-1, when in the inflammatory microenvironment, ICAM-1 MSCs significantly the up regulation of cell surface adhesion molecules, further study showed that the gene was knocked out or closed function, the inhibiting effect of MSCs immunization was significantly reduced. In previous work by our fine Cell transfection technique to improve the expression of ICAM-1 on the surface of MSCs, to construct stable MSCs high expression cell line ICAM-1. The results showed that ICAM-1 modified MSCs can play a powerful immunosuppressive function in vitro. However, ICAM-1 modified MSCs can play a role in regulating immune ability in vivo, which is not able to optimize the therapeutic effect of IBD has not been reported based on this, we establish a stable animal model, the effects of ICAM-1 modified MSCs for in vivo efficacy of IBD, and to explore its mechanism. We established the classic dextran sodium sulfate (dextran sulfate, sodium, DSS) IBD mouse model induced by DSS solution. By drinking different concentration, different concentrations of DSS solution for inflammatory bowel disease in mice model; IBD mice were treated with rat tail vein infusion of ICAM-1 MSCs modified by in vivo experiment on IBD Therapeutic effect. (1) male BALB/c mice were randomly divided into control group and 3%-DSS, 5%DSS, 7%-DSS in drinking group. The indexes were observed, on IBD mice model induced by different concentration of DSS, in order to establish a stable and repeatable high maneuverability, good model, and a firm foundation for the treatment of IBD cells lay. (2) primary cultured MSCs and C3 cells, the expression of C3 ICAM-1 cells detected by flow cytometry, and amplified in vitro and cryopreservation of cells for subsequent experiments. (3) cell therapy, experimental group: Control group, PBS group, MSCs group, C3 group, C3-MIGRI group, C3-ICAM-1 group, model third days cells of rat tail vein injection transplantation, injection volume of 500 l concentration is 5 * 106 /ml cell suspension, the frozen sections of MSCs distribution in mice, observe the general condition of mice, survival rate, target organ pathological changes detection, IBD immune factors The expression level of Th cell subsets and the proportion of 5%, 7% and 3%. Drinking, the concentration of DSS in colon formation in mice sixth days had different degrees of ulcer model was increased with DSS concentration increasing, but the mortality rate of mice also increased the concentration of 5% DSS solution in drinking contributes to efficient, economical to build IBD mouse model laid a solid foundation for the treatment of IBD. MSCs cell homing experiments showed that overexpression of ICAM-1 can significantly improve the MSCs homing efficiency, and its regulating function of tissue repair and immune, ICAM-1 modified MSCs can promote IBD mice spirit, diet, body weight and general condition of recovery activities, improve survival rate, reduce the pathological injury. Differentiation of Th cell subsets in the spleen decreased, IFN- gamma, IL-17 level, increased Foxp3 level. In conclusion, this study successfully established a mouse model of IBD induced by DSS, ICAM-1 modified MSCs can promote targeted for As well as immunosuppressive effects, the therapeutic effect of MSCs on IBD was effectively enhanced, and the field of IBD cell therapy was widened.

【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R574.62

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