發(fā)布時間：2018-03-01 07:37

  本文關鍵詞： 炎癥性腸病 間充質(zhì)干細胞 細胞間粘附分子-1 細胞治療 靶向作用　出處：《河北北方學院》2016年碩士論文　論文類型：學位論文

【摘要】：間充質(zhì)干細胞(Mesenchymal stem cells,MSCs)移植治療炎癥性腸病(inflammatory bowel diseases,IBD)已經(jīng)成為IBD治療領域有發(fā)展前途的治療策略。目前主流研究認為IBD的發(fā)病機制主要與非致病性腸抗原作用于遺傳易感者引起的免疫應答失調(diào)有關,最終引起腸道黏膜屏障功能障礙進而導致腸組織損傷。MSCs具有免疫調(diào)節(jié)和促組織修復的生物學特性,基于此,MSCs已經(jīng)被嘗試應用于治療免疫相關疾病,并且取得了良好的療效。目前研究表明MSCs能夠歸巢至受損腸道組織,有效地促進黏膜修復,緩解IBD的臨床癥狀。此外,MSCs治療IBD具有良好的安全性。因此,MSCs療法為臨床治療IBD,尤其是嚴重難治IBD提供了一個較好的選擇。但是,目前MSCs的臨床應用還存在著治療效率不高,療效不夠穩(wěn)定等不足,因此未來的研究應著眼于優(yōu)化MSCs的治療效果,提高MSCs移植治療的穩(wěn)定性等。MSCs的免疫抑制功能主要通過分泌可溶性分子和表達細胞表面粘附分子來實現(xiàn)。更重要的是,MSCs的定向遷移能力取決于配體和受體間的相互作用。細胞間粘附分子-1(Intercellular adhesion molecules 1,ICAM-1)是MSCs發(fā)揮功能的關鍵分子,生理狀態(tài)下MSCs表達較低水平的ICAM-1,當處于炎性微環(huán)境中時,MSCs顯著上調(diào)其細胞表面的ICAM-1,進一步研究表明粘附分子基因被敲除或封閉功能后,MSCs的免疫抑制作用顯著降低。我們在前期工作中通過細胞轉(zhuǎn)染技術提高MSCs表面ICAM-1表達量,構建了穩(wěn)定高表達ICAM-1的MSCs細胞株。相關研究結(jié)果顯示ICAM-1修飾的MSCs可在體外發(fā)揮強大免疫抑制功能。但是,ICAM-1修飾MSCs能否在體內(nèi)發(fā)揮免疫調(diào)節(jié)能力,其是否能夠優(yōu)化IBD的治療效果目前尚未見報道�；�于此,我們建立了穩(wěn)定的動物模型,考察ICAM-1修飾的MSCs對于IBD的體內(nèi)療效,并進一步探索其內(nèi)在機制。我們建立了經(jīng)典的葡聚糖硫酸鈉(dextran sulfate sodium,DSS)誘導的IBD小鼠模型。通過自由飲用不同濃度的DSS溶液,探討不同濃度的DSS溶液對于炎癥性腸病小鼠模型的影響;鼠尾靜脈輸注ICAM-1修飾的MSCs對IBD小鼠進行治療,通過體內(nèi)外實驗探討其對IBD的治療作用。(1)雄性BALB/c小鼠隨機分為對照組和3%-DSS,5%DSS,7%-DSS飲用組。觀察各項指標,系統(tǒng)探討不同濃度DSS誘導的IBD小鼠模型,以期建立可重復、穩(wěn)定高、可操作性優(yōu)良的模型,從而為IBD的細胞治療打下堅定的基礎。(2)體外培養(yǎng)原代MSCs及C3細胞系,應用流式細胞術檢測C3細胞系ICAM-1的表達量,并體外擴增及凍存細胞,以備后續(xù)實驗應用。(3)細胞治療,實驗分組:Control組,PBS組,MSCs組,C3組,C3-MIGRI組,C3-ICAM-1組,建模第3天進行細胞鼠尾靜脈注射移植,注射量為500μl濃度為5×106個/ml的細胞混懸液,冰凍切片觀察MSCs在小鼠體內(nèi)分布,觀察各組小鼠一般情況、生存率,檢測靶器官病理變化,IBD相關免疫因子表達水平和Th細胞亞群比例。飲用3%,5%,7%濃度DSS的小鼠在第6天結(jié)腸均有不同程度潰瘍形成,成模率隨著DSS濃度提升而增加,但是小鼠死亡率也相應增加,飲用5%濃度的DSS溶液有助于高效、經(jīng)濟地建立IBD小鼠模型,為MSCs治療IBD打下了堅定的基礎。細胞歸巢實驗顯示過表達ICAM-1可顯著提高MSCs靶向歸巢效率,進而發(fā)揮組織修復和免疫調(diào)節(jié)功能,ICAM-1修飾的MSCs可促進IBD小鼠精神、飲食、體重和活動等一般狀況恢復,提高生存率,減輕病理損傷,影響Th細胞亞群的分化,降低脾臟中IFN-γ,IL-17的水平,增加Foxp3的水平。綜上所述,本研究成功的建立了DSS誘導的IBD小鼠模型,ICAM-1修飾的MSCs可通過促靶向作用以及免疫抑制作用,有效地增強了MSCs對于IBD的治療效果,拓寬了IBD細胞治療的領域。
[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.

【學位授予單位】：河北北方學院
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R574.62

【參考文獻】

相關期刊論文 前10條

1 Ana I Flores;Gonzalo J Gómez-Gómez;ángeles Masedo-González;M Pilar Martínez-Montiel;;Stem cell therapy in inflammatory bowel disease: A promising therapeutic strategy?[J];World Journal of Stem Cells;2015年02期

2 楊旭;沈洪;陸sチ，

本文編號：1550980