Genistein联合环孢素A对大鼠心脏移植急性排斥反应中CXCR3表达的干预

发布时间：2018-03-21 15:54

本文选题：移植排斥反应　切入点：Genistein　出处：《山东大学》2015年硕士论文　论文类型：学位论文

【摘要】：背景与目的随着手术技术的不断发展、免疫抑制剂的推广应用及综合医疗水平的提高,心脏移植技术得到了快速的发展,患者移植术后的生存率也得到了明显的提升。但在实际临床中,患者仍需面临超急性排斥反应、急性排斥反应和慢性排斥反应等免疫反应的危险,且其作用直接影响患者的预后及生存,其中对急性排斥反应的早期诊断和干预治疗尤为重要。众多研究表明,趋化因子受体及配体间的相互作用,在免疫细胞的诱导活化、特异性募集和宿主抗移植物移植排斥反应中发挥着重要作用,在移植免疫的急性排斥反应中异常活跃。因此趋化因子受体的拮抗剂有望在移植免疫抑制方面发挥重要作用。趋化因子受体CXCR3是G蛋白超家族的成员,其与配体间的特异性结合可诱导以T淋巴细胞为主的多种免疫细胞的活化和增殖,是导致急性排斥反应中炎症细胞浸润和组织损伤的重要因素。如果能阻断CXCR3的活化过程,抑制相关免疫细胞的增殖,就可能对移植心脏起到保护作用。前期研究证实,CXCR3活化过程中的下游级联反应依赖酪氨酸蛋白激酶(PTK)的激活,而Genistein具有抑制酪氨酸蛋白激酶的作用。本实验通过建立大鼠急性排斥反应及免疫耐受模型,观察CXCR3与心脏移植急性排斥反应的相关性,并探讨Genistein联合环孢素A对大鼠同种异体急性排斥反应中CXCR3表达的影响,为心脏移植急性排斥反应的免疫抑制治疗提供新的方向。方法选取近交系Wistar大鼠为供者,SD大鼠为受者,使用“套管连接”技术将供者心脏移植至受者大鼠右侧颈部皮下。移植后的大鼠随机分为3组,每组12只,急性排斥反应(AR)组：心脏移植术后未采取任何治疗；环孢素A(CsA)组：受者鼠移植术后接受CsA 10mg/kg/d治疗；CsA+Genistein (C+G)组：术后接受CsA 10mg/kg/d及Genistein lOmg/kg/d的联合治疗。术后每天通过视诊或触诊受者鼠右侧颈部,观察移植心脏存活情况。术后第7天,采集3组移植心脏,制备成组织切片,HE染色观察移植心脏的病理学改变,免疫组织化学染色及Western blotting法检钡CXCR3受体的表达,采用图像分析软件对其表达程度进行半定量分析。结果CsA组和C+G组的移植心脏存活的情况要明显好于AR组,移植心脏免疫耐受性良好。HE染色结果可见,AR组中的移植心脏组织可见的弥漫的炎性细胞浸润和心肌细胞变性坏死,呈现急性排斥反应的典型表现,C+G组的排斥反应强度明显弱于前两组。免疫组化染色中CXCR3在各组中的IOD值为：AR组(1.05±0.20)×105,CsA组(0.53±0.16)×105,C+G组(0.24±0.08)×105。CsA组的表达水平明显低于AR组；C+G组的表达水平明显低于前2组(P0.05)。Western blotting中检测各组的灰度比值的趋势与免疫组化检测结果一致,C+G组移植心脏组织中CXCR3蛋白的表达水平明显低于前2组(P0.05)。结论CXCR3的表达程度与心脏移植急性排斥反应的程度呈正相关；Genistein联合CsA可明显减轻移植心脏组织中CXCR3的表达,能进一步减轻急性排斥反应。
[Abstract]:Background and objective with the development of surgical techniques, immunosuppression and application of comprehensive health care level, heart transplantation technology has been rapid development, the survival rate of patients after transplantation has been significantly improved. But in clinical practice, patients still need to face the danger of hyperacute rejection, acute rejection chronic rejection and immune response, and its effect directly affect the prognosis and survival of patients, the early diagnosis and treatment of acute allograft rejection is very important. Many studies have shown that chemokine receptor interactions and ligand, in induced activation of immune cells, plays an important role in raising specific and host versus graft rejection, very active in the acute rejection of transplantation immune reaction. Therefore chemokine receptor antagonists in transplantation immune suppression is expected to Play an important role. The chemokine receptor CXCR3 is a member of the G protein superfamily, which is combined with the specific ligand can induce a variety of immune cells in the T lymphocyte activation and proliferation, is an important factor leading to inflammatory cell infiltration and tissue injury in acute rejection. If we can block the activation process of CXCR3, related to the inhibition of the proliferation of immune cells. It may protect the heart transplantation. Previous studies demonstrated that the downstream cascade activation of CXCR3 dependent protein tyrosine kinase (PTK) activation, and Genistein can inhibit the role of protein tyrosine kinase. This experiment establish and acute rejection in rat model of immune tolerance correlation, observation of CXCR3 and acute rejection of heart transplantation, and investigate the effect of Genistein combined with cyclosporine A on the expression of CXCR3 in rat acute allograft rejection effect, Immune to acute rejection of heart transplantation rejection and provide a new direction of treatment. Methods of inbred Wistar rats as donors and SD rats as recipients, the use of "connection" technology of the donor heart transplantation to recipient rats subcutaneously on the right side of the neck. The rats were randomly divided into 3 groups after transplantation, 12 rats in each group, acute rejection (AR) group: no treatment after heart transplantation; cyclosporine A (CsA) group: transplantation rats underwent CsA after 10mg/kg/d treatment; CsA+Genistein (C+G) group: after receiving combination therapy and Genistein lOmg/kg/d CsA 10mg/kg/d. Every day after operation by visual examination or palpation of recipients on the right side of the neck, to observe the cardiac allograft survival. After seventh days, collecting 3 groups of heart transplantation, preparation of tissue sections to observe the pathological changes of the transplanted heart HE staining, immunohistochemical staining and Western blotting method for detecting CXCR3 receptor The expression of image analysis software was used for semi quantitative analysis of the degree of expression. Results CsA group and C+G group of cardiac allograft survival is significantly better in the AR group, heart transplantation immune tolerance.HE good dyeing result shows that in the AR group of heart tissue transplantation visible diffuse infiltration of inflammatory cells and myocardial cells the typical performance showed degeneration and necrosis, acute rejection, rejection of the strength of C+G group was significantly lower than the former two groups. Immunohistochemical staining of CXCR3 in each group of the IOD value is: AR group (1.05 + 0.20) * 105, CsA group (0.53 + 0.16) * 105, C+G group (0.24 + 0.08) * 105.CsA group the expression level was significantly lower than that of AR group; the expression level of C+G group was significantly lower than the former 2 groups (P0.05) were detected in.Western gray level ratio in the blotting trend and immunohistochemical detection results, CXCR3 protein C+G in heart tissue transplantation group was significantly lower In the first 2 groups (P0.05). Conclusion the degree of CXCR3 expression is positively correlated with the degree of acute rejection in heart transplantation. Genistein combined with CsA can significantly reduce the expression of CXCR3 in transplanted heart tissue, and further reduce the acute rejection.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R654.2

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