UC-MSCs诱导表达IDO促烧伤大鼠修复的机制研究

发布时间：2018-01-20 12:24

本文关键词： UC-MSCs IDO 免疫调节烧伤炎症　出处：《新疆医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:比较体外诱导表达吲哚2,3-双加氧酶(indoleamine2,3-dioxygenase,IDO)的脐带间充质干细胞)对T细胞增殖能力的影响,了解IDO高表达的UC-MSCs移植治疗大鼠烧伤的免疫调节机制、促烧伤修复效果,探讨UC-MSCs在烧伤治疗的临床应用前景。方法:(1)体外实验:建立稳定的UC-MSCs系,用干扰素γ(Interferon-γ,IFN-γ)诱导UC-MSCs产生IDO,并分别用逆转录聚合链式反应(Reverse Transcription Polymerase Chain Reaction RT-PCR)和Western Blot检测诱导后UC-MSCs的IDO信使核糖核酸(Messenger ribonucleic acid,mRNA)表达水平和蛋白表达水平;将T细胞分别与不同比例及不同组别的UC-MSCs共培养,检测比较培养体系中T细胞的增殖能力。(2)动物实验:取体重200±5g SD(sprague-dawley)雄性大鼠294只随机分为预实验组、盐水对照组、空白对照组、MSCs组、Induced MSCs组、Induced MSCs+1-MT组。根据预实验组烧伤后炎症介质变化曲线,确定UC-MSCs移植时间点。将盐水对照组、空白对照组、MSCs组、Induced MSCs组、Induced MSCs+1-MT组共计240只大鼠制备烧伤模型,在烧伤24h后对盐水对照组进行皮下盐水注射,MSCs组进行UC-MSCs移植,Induced MSCs组移植经IFN-γ诱导表达IDO的UC-MSCs、Induced MSCs+1-MT组移植IFN-γ诱导表达IDO的UC-MSCs并添加IDO的拮抗剂1-MT,以上UC-MSCs的移植量均为2×106。并在烧伤后0d、1d、2d、3d、5d、7d、9d采集血样,检测其WBC、CRP及炎症因子IFN-γ、TNF-α、IL-6、IL-10表达变化,通过图像分析软件比较14d、21d、28d时各组创面愈合率。结果:(1)通过RT-PCR和Western Blot检测,结果显示未诱导前UC-MSCs的IDO表达较弱,经过IFN-γ诱导后,其IDO的表达增强,且随着IFN-γ浓度的增加,UC-MSCs的IDO表达呈现出逐渐增强的趋势。(2)共培养体系中,诱导产生IDO的UC-MSCs对T淋巴细胞的增殖与其他各组相比具有明显的抑制作用(P0.01),未经诱导的UC-MSCs组对T细胞的增殖抑制作用强于IFN-γ诱导后添加1-MT拮抗剂的UC-MSCs移植组,相同条件下加入IDO特异性的抑制剂1-MT的共培养体系中,UC-MSCs对T淋巴细胞的增殖抑制作用轻微。(3)在烧伤模型的炎症因子检测中,炎症因子IFN-γ、TNF-α、IL-6、IL-10在烧伤后七天内出现不同程度的升高。Induced MSCs组和MSCs组较两个对照组及诱导后MSCs+1-MT组的WBC、CRP和炎症因子TNF-α、IL-6、IL-10各项指标均有不同程度的降低,但Induced MSCs组的炎症指标较MSCs组下降幅度更为显著,IFN-γ水平未见显著差别。(4)创面愈合率。烧伤后14d,MSCs组、Induced MSCs组、Induced MSCs+1-MT组与两组对照组相比,创面愈合率较高,但三组之间无统计学差异;烧伤后21d和28d,Induced MSCs组、MSCs组、Induced MSCs+1-MT组的创面愈合率显著高于两个对照组,且三组之间存在差异,即Induced MSCs组优于MSCs组和Induced MSCs+1-MT组,而MSCs组优于Induced MSCs+1-MT组。结论:经IFN-γ诱导表达IDO的UC-MSCs对T淋巴细胞的增殖有明显抑制作用。通过烧伤后移植UC-MSCs可降低烧伤后炎症相关指标,而经IFN-γ诱导后的UC-MSCs对炎症免疫应答水平的调节作用更为显著,IDO是UC-MSCs发挥抑制炎症作用的关键分子之一。在烧伤大鼠的组织修复过程中,UC-MSCs移植可促进损伤部位血管形成、成纤维细胞的增殖以及后续的皮肤修复重建,促进烧伤创面愈合,IDO参与了这一免疫调节作用且发挥重要作用。
[Abstract]:Objective: To compare the in vitro expression of 2,3- indole dioxygenase (indoleamine2,3-dioxygenase, IDO) of human umbilical cord mesenchymal stem cells) effect on T cell proliferation, immune to high expression of IDO UC-MSCs transplantation in the treatment of burn rats with adjustment mechanism, promote the burn repair effect of UC-MSCs in clinical application in the treatment of burn injury. Methods: (1) in vitro: to establish a stable UC-MSCs system with interferon gamma (Interferon- y, IFN- y) induced UC-MSCs production and IDO, respectively by reverse transcription polymerase chain reaction (Reverse Transcription Polymerase Chain Reaction RT-PCR) and Western Blot detected UC-MSCs IDO messenger RNA (Messenger ribonucleic, acid, mRNA) expression the expression level and protein; T cells were UC-MSCs with different proportion and different groups of co cultured T cell culture system, detection and comparison of proliferation ability (2) animal. Results: the weight of 200 + 5g SD (Sprague-Dawley) male rats 294 were randomly divided into pre experimental group, saline control group, blank control group, MSCs group, Induced MSCs group, Induced MSCs+1-MT group. According to the curve changes of inflammatory mediators pre experimental group after burn, UC-MSCs transplantation time points. The saline control group and the blank control group, MSCs group, Induced MSCs group, Induced MSCs+1-MT group consisted of 240 rats in model group were prepared by burn, subcutaneous saline injection of saline in burn after 24h, MSCs group UC-MSCs Induced group MSCs after transplantation, transplantation of IFN- gamma induced expression of IDO UC-MSCs and 1-MT Induced antagonist MSCs+1-MT group transplantation of IFN- gamma induced expression of IDO UC-MSCs and adding IDO, transplantation above UC-MSCs were 2 * 106. and after burn 0d, 1D, 2D, 3D, 5D, 7d, 9D, blood samples were collected to detect the WBC, CRP and inflammatory factor IFN- gamma, TNF- alpha, IL-6, expression of IL-10, through the figure Image analysis software for comparison of 14d, 21d, 28d of the group wound healing rate. Results: (1) by RT-PCR and Western Blot detection results showed no UC-MSCs before induction of IDO expression is weak, after induced by IFN-, increase the expression of IDO, and with the increase of IFN- gamma concentration, UC-MSCs showed IDO expression the trend of a gradual increase. (2) co culture system, IDO induced UC-MSCs proliferation of T lymphocytes and the other groups have obvious inhibitory effect (P0.01), adding UC-MSCs transplantation group 1-MT antagonist group without UC-MSCs induced proliferation of T cells inhibited by IFN- gamma induced after under the same conditions, adding IDO specific inhibitors of 1-MT co culture system, UC-MSCs on proliferation of T lymphocytes inhibited. (3) in the detection of inflammatory cytokines in burn model, inflammatory factor IFN- gamma, alpha TNF-, IL-6, IL-10 in seven days after burn in Different degrees of elevated.Induced MSCs group and MSCs group compared with the control group two and group MSCs+1-MT after induction of WBC, CRP and inflammatory factor TNF- alpha, IL-6, lower IL-10 indicators have different degrees of inflammation, but the index of Induced MSCs group than in MSCs group decreased more significantly, IFN- levels were no significant difference. (4) the rate of wound healing after burn. 14d, MSCs group, Induced MSCs group, Induced MSCs+1-MT group compared with two control groups, the wound healing rate is high, but there is no significant difference between the three groups; after burn 21d and 28d, Induced, MSCs group, MSCs group, Induced MSCs+1-MT group wound healing rate was significantly higher than that in two a control group, and the differences between the three groups, namely Induced MSCs group than in MSCs group and Induced MSCs+1-MT group, and MSCs group than in Induced group. Conclusion: MSCs+1-MT can significantly inhibit the expression of IDO by IFN- induced UC-MSCs proliferation of T lymphocytes. Burn after transplantation of UC-MSCs can reduce the inflammation related indicators after burn, while the IFN- gamma induced UC-MSCs on inflammatory immune response regulation is more significant, IDO UC-MSCs is one of the key molecules play inhibit inflammation. In the repair of burn rat tissues, UC-MSCs transplantation can promote the formation of vascular injury. A subsequent reconstruction and repair of skin fibroblast proliferation, promote wound healing, IDO are involved in the immune regulation and play an important role.

【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R644

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