【作者】 李伟栋；

【导师】 倪一鸣；

【作者基本信息】 浙江大学 ， 外科学， 2004， 硕士

【摘要】 前言 终末期器官功能衰竭病人行常规治疗无效时，器官移植是重要的治疗办法之一。器官移植的主要障碍是同种异体排斥反应。它是一种特异性的细胞免疫应答，其实质和关键是T细胞活化。而辅助性T淋巴细胞(Th细胞)的活化、增殖和分化是免疫应答反应阶段中的第一步，Th细胞活化后增殖并分化为效应Th细胞而分泌多种细胞因子，这些细胞因子在之后的免疫应答中起到了重要的介质作用，进一步诱导效应性T细胞活化、增殖和分化而迅速扩大免疫排斥效应。根据介导免疫反应类型和作用的不同，可将细胞因子分为两类：Th1和Th2。 白介素10(IL-10)是一种Th2类因子，大量的研究发现它具有抑制APC表面MHCII类分子的表达，抑制Th1细胞合成细胞因子，抑制CTL细胞和NK细胞活性等作用。在器官移植领域中，研究发现一定时期内的免疫抑制与IL-10的高表达存在着密切的正相关，IL-10对移植物排斥反应起到了一种明显的抑制作用。 本研究应用小鼠腹腔内异位心脏移植模型，通过质粒载体将外源性IL-10基因转染进移植心，通过应用病理学、RT-PCR、免疫组化等免疫学及分子生物学实验技术手段，观察质粒载体在移植心内的转染效率和基因的表达效率。并探索IL-10分子在同种异体移植物急性排斥反应中的表达情况及其意义。同时探索移植心局部IL-10的高表达能否有效地抑制异基因小鼠心脏移植排斥反应及其可能的作用机制。 材料与方法 C57BL／6和Balb／c小鼠作为移植供体，Balb／c小鼠作为移植受体，建立小鼠腹腔内异位心脏浙江大学硕士学位论文移植模型。按移植前供心处理方法不同，随机分为5组:A组为Baib/c一Balb/c同基因对照组;B组为C57BL/6一Balb/c+NS;C组为C57BL16·Balb/c+PO盯一xT七;D组为c57BL/6一Balb/c+poRF一mIL一10;E组为C57BL/6一Balb/c+PO灯·ITRs一mIL一10;C、D、E各组供心移植前经升主动脉冠脉灌注30ul相应质粒与脂质体的混合物;B组用等量生理盐水。移植后l、3、5、7天切取移植心脏用于病理学检测、浸润淋巴细胞的免疫组化、移植心肌组织中基因产物的逆转录聚合酶链式反应和IL一10的免疫组化检测;另外设亚组观察移植后一般情况和移植心存活时间。 实验所得数据以王士s表示，统计学处理用spssl2.0软件完成.各组受体移植心存活率评估以Kaplan书eier法检验，组间存活时间的比较以非参数Mann WhitneyU检验进行，其它参数作one一ay AN0vA分析及LSD检验，P<0.05表示有差异，P<0 .01表示有显著性差异。 结果 A组小鼠及移植心均获长期存活(大于100天)，B组和C组移植心存活时间分别为8.125士0.991天和7.714士0.756天，D组和E组移植心存活时间分别为15.714士2.498天和17.857士1.864天，与B组及C比较，D和E组的移植心存活时间明显延长。和D组相比，E移植心存活时间也明显延长。A组各个时间段移植心病理学检查均未发现排斥现象，B组和C组小鼠术后第3天起心肌间淋巴细胞浸润明显，心肌细胞肿胀坏死，术后第5、7天尤甚。D和E组小鼠术后3、5、7天心肌间淋巴细胞浸润程度及心肌细胞坏死程度较B组和C组明显减轻。移植心内基因产物RT一PcR结果显示，A组各时间段移植心标本内均可检测到IL一10 mRNA的弱表达。B组和C组移植后第1、3天可见IL一10 mRNA弱表达，在其后第5、7天均无表达。而D和E组在各个时间段都可见明显的IL一10 mRNA的阳性表达，3天上升，5天达到高峰，7天下降。第3、5、7天相对表达值均显著高于B组和C组。而E组第3、5、7天移植心内标本中IL一10 mRNA相对表达值也明显高于D组。各组IL一10蛋白免疫组化检测也发现了相似的IL一10分子表达规律。 浸润淋巴细胞的免疫组化分型发现，A组移植后各时间点移植心肌组织中有少量CO4+和CDS+浸润细胞。而B组和C组移植后第3天起，CD4干和CDS+T细胞数量逐渐增多，5天达到高峰，并延续至第7天。组内各时间点纵向比较发现，心肌组织中浸润CD4+T细胞量在第3天明显增加，而cDS+T细胞数量至第5天才明显增加，D组和E组移植后移植心浸润cD4+T细胞和CDS+T细胞有缓慢地增加，但在第3、5、7天CD4+T细胞的量和第5、7天，CDS+T细胞的量比B组和C组明显减少，而与D组相比，E组移植后第3天移植心浸润CD矿T细胞数和移植后第5、7天移植心肌组织中浸润CDS+T细胞数明显减少。浙江大学医学院附属第一医院 浙江大学硕士学位论文 讨论 D组和E组第3、5、7天移植心内标本中IL一10 mRNA相对表达值均明显高于B和C组，而E组第3、5、7天移植心内标本中IL一10 mRNA相对表达值也明显高于D组，心肌细胞内IL一10分子免疫组化检测也发现了相同表达规律，这说明在本实验条件下，p0RF--mIL一10质粒能成功地转染心肌细胞并使mIL一10进行持久大量的表达。而ITRs可以明显提高p0RF一ITRs--mIL一10质粒在心肌细胞中的转染效率和mIL一10表达水平。 移植心内IL一10 mRNA和IL一10分子的的表达规律，与移植心组织病理学结果恰好相反。而外源性IL一10基因能?更多还原

【Abstract】 IntroductionOrgan transplantation has become a well-established modality for treatment of end stage disease.Allograft rejection will still be the main cause of death in transplant recipients who survived an indefinite time. Allograft rejection is a specific cellular immuneresponse mainly due to T cell activation. While the activation of the help T cells(Th cell) plays a critical role in the response stage. The activated Th cells can be transformed into effective Th cells and secret lots of cytokine which act as an important medium in the following immunoresponse stage. They induce the activation,proliferation and differentiation of effective lymphocytes to proceed allograft rejection. These cytokines can be divided into two groups, Thl and Th2, according to its effect on the allograft immunoresponse.Interleukin 10(IL-10) is a kind of Th2 cytokine. Previous researchs have demonstrated that IL-10 can reduce the expression of major histocompatibility complex II (MHCII) molecule on the surface of antigen-presenting cell(APC), decrease the secretion of Thl cytokin, inhibit the activation of cytotoxic T cells(CTL), macrophages and NK cells. Furthermore, in the organ transplantation field, recent reports have confirmed that the localized immunosuppression in the allograft is parellel to expression level of IL-10. The high expression of IL-10 can act an obvious immunosuppression of allograft rejection.In the present research we transfer IL-10 gene into cardiomyocytes in transplanted heart in mice and investigated gene transfer efficiency and expression in allograft as well as the intrinsic IL-10 expression and its implication in allo-immune response by means of pathology, RT-PCR, immunohistochemical staining. We also explored whether extrinsic IL-10 is able to inhibit the allograft rejection and their possible mechanism.Materials and MethodsA total of 200 mice (donors and recipients) were used in our experiments. Inbred female Balb/c (H-2d) mice were used as syngeneic donors and C57BL/6(H-2b) were used as allogeneic donors. All recipients were Balb/c (H-2d) mice who were randomly divided as follows: Group A: Syngeneic control (Balb/c-Balb/c) ;Group B: C57BL/6-Balb/c+NS; Group C: C57BL/6-Balb/c+pORF-ITRs; Group D: C57BL/6-Balb/c+pORF-mIL-10; Group E: C57BL/6-Balb/c+pORF-ITRs-mIL-10. All donors of group D, E, F were treated with 30ul plasmid/lipofectin complex by coronary perfusion through the ascending aorta before tranlplantation and donars of group B treated with 30ul NS. All groups were subdivided into day 1, 3, 5, 7 posttransplantation respectively for sample harvesting, and additional subgroups for observation of general situation and survival time. Graft specimens were harvested to determine morphological changes by pathological examination and gene expression by reverse transcription-polymerase chain reaction (RT-PCR), as well as protein expression by immunohistochemical staining. The graft infiltrated T cell subsets were also analyzed by immunohistochemical staining.All data were expressed as mean SD. Statistical analysis was performed by SPSS 12.0 software. Kaplan-Meier was used to analyse the survival rate between each groups Mann Whitney U test was used to determine the difference of graft survival time between each groups. One-way ANOVA and LSD Test were also used for parametric data analysis between groups, respectively. Probability P values <0.05 were considered different and P values <0.01 significantly different.ResultsAll transplanted hearts of group A survived more than 100 days. The mean survival time of transplanted hearts in group B and C was 8. 125 + 0. 991 days and 7. 715+0. 756 days respectively. Prolonged survival time of transplanted hearts were found within group D and group E as compared to that in group B and group C (15. 714+2.498 days and 17.857 + 1.864 days, respectively). The similar results were also found within group E as compared to that in group D. No signs of rejection were found at any time point in group A with minimal myocardial inflammatory infiltratio更多还原