G-CSF对同种异体大鼠心脏移植的抗排斥反应作用及其机理研究

发布时间：2018-07-11 11:08

本文选题：粒细胞集落刺激因子 + CD4+CD25+调节性T细胞　；参考：《第四军医大学》2008年硕士论文

【摘要】： 自Barnard于1967年首次成功完成人类心脏移植手术以来,经过近半个世纪的发展,目前同种异体心脏移植被公认为是治疗各种终末期心脏病的最有效方法,并广泛地应用于临床。然而移植排斥反应始终是困扰临床医生的一个主要问题。粒细胞集落刺激因子(G-CSF)是最重要的造血生长因子之一,最新的研究成果表明其对获得性免疫和T细胞耐受有十分重要的作用,但机制尚未阐明。本研究拟应用改良的同种异体大鼠腹腔异位心脏移植模型,探讨G-CSF对心脏移植免疫的调节作用及其可能机制。目的:改良原有大鼠腹腔异位心脏移植模型的外科技巧,建立成功率高且更加易于实施的心脏移植模型。利用此改良模型研究G-CSF对心脏移植排斥反应的作用,探讨其可能机理,为临床有效调控、治疗心脏移植排斥反应及其他免疫性疾病提供理论依据。方法:1.对传统Ono术式的受体准备、供心切取和吻合方法等环节进行改进,采用此改良方法建立大鼠腹部异位心脏移植模型。2. Lewis(RT~1)与BN(RT~n)大鼠各40只随机分为4组(每组10只,Lewis大鼠为受体,BN大鼠为供体),于移植术后当天起分别给予各组受体鼠重组人粒细胞集落刺激因子(rh-G-CSF)0μg/kg/day(对照组)、125μg/kg/day、250μg/kg/day、500μg/kg/day,共6天。术后第6天行供心组织学检查、混合淋巴反应实验(MLR),并观察各组供心存活时间。探讨不同剂量的rh-G-CSF对大鼠心脏移植排斥反应的抑制作用。3.对于第二步实验筛选出的250μg/kg/day组进一步行细胞因子检测、外周血流式细胞仪检测以及过继转移实验,其各项指标与各对照组(包括普通大鼠、只移植心脏不使用G-CSF大鼠、只使用G-CSF不移植心脏大鼠)比较,探讨rh-G-CSF对心脏移植排斥反应抑制作用的机理。结果:1.共实施100例大鼠腹部异位心脏移植术,89例成功。供心缺血时间32±5 min,整个手术耗时60±10 min。2. 250μg组与500μg组在体内、外均显示排斥反应较对照组显著减弱,125μg组中以上各指标与对照组比较无显著差异。3.在心脏移植受体鼠和普通大鼠的骨髓中CD4+CD25+T细胞比例显著高于外周血。在不使用rh-G-CSF的情况下,心脏移植手术前、后大鼠外周血CD4+CD25+T细胞比例无显著变化。给予普通大鼠rh-G-CSF 250μg/kg/day共6天后以及术后给予心脏移植受体鼠rh-G-CSF 250μg/kg/day共6天后,其骨髓细胞中CD4+CD25+T细胞数量都有显著地下降,并伴随外周血CD4+CD25+T细胞比例显著增加。Rh-G-CSF处理心脏移植受体鼠后,使其MLR体系中白细胞介素10(IL-10)、转化生长因子?1(TGF-?1)显著增加,其包含有CD4+CD25+T细胞的脾细胞可以将抗排斥反应作用过继转移给第二级受体鼠,并且在过继转移实验和MLR中均显示出较强的免疫抑制能力。结论:1.经典的Ono术式经改进后,术中缩短了供心缺血时间,降低了吻合难度,手术成功率高。整个过程均可单人操作,无需显微镜,使其易于掌握与开展。2. G-CSF可以在体内、外诱导T细胞的低反应性,其抗心脏移植排斥反应的作用与剂量有关。3.同种异体抗原刺激和手术创伤并不能诱导CD4+CD25+调节性T细胞的增殖。G-CSF增加外周血的CD4+CD25+T细胞是其发挥抗排斥作用的重要机制。G-CSF将骨髓中的CD4+CD25+T细胞动员至外周血是本实验外周血中CD4+CD25+T细胞增加的主要机理。并且同种异体抗原的刺激提升了CD4+CD25+T细胞在体内、外抑制潜能。其中IL-10和TGF-β1是发挥免疫抑制作用的重要效应细胞因子。
[Abstract]:Since the first successful completion of human heart transplantation by Barnard in 1967, after nearly half a century of development, allograft heart transplantation is now recognized as the most effective method for the treatment of various end-stage heart diseases and is widely used in clinical practice. However, graft rejection is a major problem that puzzles clinicians. Cell colony stimulating factor (G-CSF) is one of the most important hematopoietic growth factors. The latest research results show that it has a very important role in acquired immunity and T cell tolerance, but the mechanism has not been elucidated. This study is intended to apply a modified heterotopic heart transplant model of allogeneic rat intraperitoneal and to explore the regulation of G-CSF on heart transplantation immunity. The role and its possible mechanism.
Objective: to improve the surgical skills of the original rat model of heterotopic heart transplantation, to establish a heart transplant model with high success rate and more easy to implement. This modified model is used to study the effect of G-CSF on rejection of heart transplantation and to explore its possible mechanism for clinical effective regulation and treatment of heart transplant rejection and other immune diseases. Provide a theoretical basis.
Methods: 1. to prepare for the traditional Ono procedure, for the improvement of the heart cutting and anastomosis, the improved method was used to establish the rat abdominal heterotopic heart transplantation model,.2. Lewis (RT~1) and BN (RT~n) rats, each of which were randomly divided into 4 groups (10 rats in each group, Lewis rats as receptors, BN rats as donor), and respectively to the day after transplantation. The recombinant human granulocyte colony stimulating factor (rh-G-CSF) 0 mu g/kg/day (control group), 125 g/kg/day, 250 g/kg/day, 500 mu g/kg/day were given for 6 days. The donor heart tissue examination, mixed lymphoid reaction test (MLR) were performed on the sixth days after operation, and the survival time of each group was observed. The rejection reaction of different doses of rh-G-CSF on rat heart transplantation was discussed. The inhibitory effects of.3. on further cell factor detection, peripheral blood flow cytometry and adoptive transfer test, selected by the second step experiment, were compared with the control group (including the common rats, only the heart transplant without G-CSF rats, only the G-CSF non transplant rat), and the study of rh-G-CSF. The mechanism of inhibition of rejection in heart transplantation.
Results: 1. 100 cases of abdominal heterotopic heart transplantation in rats were performed, 89 cases were successful. The ischemic time of donor heart was 32 + 5 min, the whole operation took 60 + 10 min.2. 250 mu g group and 500 g group in the body, and the rejection reaction was significantly lower than that of the control group. There was no significant difference between the indexes of the 125 mu g group and the contrast group.3. in the heart transplant recipient mice. The proportion of CD4+CD25+T cells in the bone marrow of the normal rats was significantly higher than that in the peripheral blood. Without the use of rh-G-CSF, there was no significant change in the proportion of CD4+CD25+T cells in the peripheral blood of the rats before the heart transplant operation. The total rh-G-CSF 250 mu g/kg/day was given to the normal rats for 6 days and the rh-G-CSF 250 mu g/kg/day was given to the heart transplant recipient rats after the operation. After the day, the number of CD4+CD25+T cells in the bone marrow cells decreased significantly, and the proportion of CD4+CD25+T cells in the peripheral blood increased significantly with the.Rh-G-CSF treatment of the heart transplant recipient rats, making the MLR system interleukin 10 (IL-10), transforming growth factor 1 (TGF-? 1) increased significantly, and the spleen cells containing CD4+CD25+T cells could be resistant to the spleen cells containing CD4+CD25+T cells. The adoptive transfer of the rejection reaction was transferred to the second level receptor mice, and both showed strong immunosuppression in adoptive transfer experiments and MLR.
Conclusion: 1. the classical Ono method has been improved, the ischemia time of donor heart is shortened, the difficulty of the anastomosis is reduced, the success rate of the operation is high. The whole process can be operated in a single person without a microscope, so that it can easily master and develop the low reverse stress of the T cells in the body, and the effect of the.2. G-CSF on the rejection of the heart transplantation and the dose. .3. allogenic antigen stimulation and surgical trauma can not induce the proliferation of CD4+CD25+ regulatory T cells and.G-CSF increase the CD4+CD25+T cells in peripheral blood, which plays an important role in anti rejection..G-CSF to mobilize CD4+CD25+T cells from the bone marrow to peripheral blood is the main mechanism of the increase of CD4+CD25+T cells in the peripheral blood of this experiment. The stimulation of allogenic antigen enhances the inhibitory potential of CD4+CD25+T cells in vivo and in vitro. Among them, IL-10 and TGF- beta 1 are important cytokines that play the role of immunosuppression.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392;R654.2

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