补体C5a对肾移植排斥反应中IL-17产生的调节作用

发布时间：2019-02-13 11:52

【摘要】：近年来，我国每年由于各种原因导致的新增肾功能衰竭病例约200万人，虽然肾移植是治疗终末期肾功能衰竭的理想方法，而能否长期维持移植肾有功能的存活，仍然是亟待解决的重要问题。目前国内外，临床上普遍采用环孢素A、他克莫司及雷帕霉素等免疫抑制剂作为肾移植术后抑制排斥反应发生的药物。这些抗排斥药物的作用机理均是通过非特异性地抑制T细胞的活化和增殖，来控制排斥反应的发生。虽然在临床上取得了明显的效果，但由于是对所有T细胞功能的全面抑制，作用缺乏选择性，副作用大。 IL-17由CD4+T亚群、CD8+T亚群、细胞和中性粒细胞等多种细胞分泌，为重要的促炎因子，可诱导人表皮细胞、内皮细胞和成纤维细胞分泌IL-6、IL-8和G-CSF；促进人成纤维细胞表达ICAM-1；募集中性粒细胞和促进血管生成。现有研究认为IL-17在器官移植术后急性排斥反应中有着重要作用。在移植排斥反应中，移植抗原与抗体的结合进一步激活补体系统。作为补体系统的枢纽分子，C5被激活后，可裂解形成C5a和C5b。C5a是一种重要的炎症介质和趋化因子，通过与靶细胞表面C5aR的结合，发挥其生物学效应。尽管补体在移植排斥中的重要作用已有不少报道，但对于小分子C5a在临床移植排斥中的作用机制并不清楚。目的：探讨肾脏移植排斥反应发生中，白细胞介素-17与补体C5活化产物C5a的表达，以及两者之间可能的相互调节作用。方法： ①首先通过流式细胞术(flow cytometry, FCM)比较同种异基因肾移植手术前后患者外周血细胞中IL-17+T细胞的百分率。 ②采用酶联免疫吸附试验(Enzyme Linked Immunosorbent Assay, ELISA)比较同种异基因肾移植手术前后患者血清补体C5a的水平。 ③采用免疫组织化学法比较发生排斥反应肾组织与正常肾组织中，IL-17的表达和补体活化产物C5b-9的沉积情况。 ④免疫细胞化学法检测人肾小管上皮细胞系HK2细胞C5aR的表达。 ⑤免疫细胞化学法和流式细胞术比较重组人C5a诱导前后，人肾小管上皮细胞系HK2细胞中IL-17产生的差异。结果： ①通过流式细胞技术检测肾脏移植前后患者外周血中IL-17+T细胞百分率，发现接受同种异基因肾脏移植患者外周血标本中，部分患者术后3天IL-17+T细胞百分率较术前明显升高。 ②ELISA结果显示，接受同种异基因肾移植术后7天时，患者血清C5a水平较术前有显著上升。 ③与正常肾脏组织相比，发生排斥反应肾脏组织IL-17的表达与补体活化产物C5b-9的沉积均明显增加，且两者的表达方式相似，均主要集中于肾小管区域。 ④免疫细胞化学结果显示，人肾小管上皮细胞系HK2细胞固有性表达C5aR。 ⑤重组人C5a刺激HK2细胞48h后，IL-17的表达水平明显上调，表明C5a可通过与固有性表达在肾小管上皮细胞上的C5aR结合，直接作用于肾小管上皮细胞调节其IL-17的产生。结论：移植排斥反应中，补体C5的活化产物C5a可通过与肾小管上皮细胞上C5aR结合，正向调节肾小管上皮细胞IL-17的产生，进而促进同种异基因移植排斥反应的发生。该研究结果可为补体抑制剂治疗移植相关疾病的使用以及发展选择性的T细胞抑制药物提供进一步的理论依据。
[Abstract]:In recent years, there are about 2 million new cases of renal failure due to various causes, although kidney transplantation is an ideal method for the treatment of end-stage renal failure, and the long-term maintenance of the function of the transplanted kidney is an important problem to be solved. At present, at home and abroad, the clinical application of the immunosuppressants such as ciclosporin A, tamosmin and rapamycin as a drug for inhibiting the rejection after renal transplantation The mechanism of action of these anti-rejection drugs is to control the generation of rejection by non-specific inhibition of the activation and proliferation of T-cells. has a clear effect on all T cell functions, a lack of selectivity, side effects, Large. IL-17 is secreted by various cells such as CD4 + T subpopulations, CD8 + T subpopulations, cells and neutrophils. It is an important pro-inflammatory factor that can induce human epidermal cells, endothelial cells and fibroblasts to secrete IL-6, IL-8 and G-CSF, promote the expression of ICAM-1 in human fibroblasts, and raise neutrophils and promote blood. The results suggest that IL-17 has an important role in the acute rejection of organ transplantation. To act. In the graft rejection, the combination of the graft antigen and the antibody is further induced The active complement system. As the hub molecule of the complement system, C5 is activated, and can be cleaved to form C5a and C5b. C5a is an important inflammatory mediator and a chemokine that will play its role by binding to the target cell surface C5aR. Biological effects. Although the important role of complement in transplant rejection has been reported, the role of small molecule C5a in clinical transplant rejection the system does not Objective: To study the expression of interleukin-17 and complement C5 activation product C5a in the development of renal transplant rejection. possible Methods: The peripheral blood cells of the patients before and after renal transplantation were compared by flow cytometry (FCM) and flow cytometry (FCM). The percentage of IL-17 + T cells was determined by enzyme-linked immunosorbent assay (ELISA). The level of serum complement C5a in the patients before and after the operation. and the deposition of the complement activation product C5b-9. Expression of C5aR in small-tube epithelial cell line HK2 cells. Human renal tubular cells were compared with the recombinant human C5a before and after C5a induction by immunohistochemistry and flow cytometry. epithelioma The results showed that the percentage of IL-17 + T cells in peripheral blood of patients before and after kidney transplantation was detected by flow cytometry. The percentage of IL-17 + T cells increased significantly in 3 days after the operation. The results of the ELISA showed that the IL-17 + T cells received the same allogenic group. The level of C5a of the patient was significantly higher than that of the normal renal tissue at 7 days after renal transplantation. The expression of IL-17 in the renal tissue of the rejection reaction and the deposition of the complement activation product C5b-9 were compared with the normal renal tissue. The product is obviously increased, and the expression of the two is similar, and is mainly concentrated in the renal tubular region. The results of the cytochemistry showed that the expression of IL-17 was up-regulated after the HK2 cells were stimulated by C5a in the human renal tubular epithelial cell line HK2. The expression of IL-17 was up-regulated after the HK2 cells were stimulated by the recombinant human C5a. supracellular Conclusion: The C5a of the complement C5 can be combined with the C5aR on the renal tubular epithelial cells by the combination of the C5aR and directly on the renal tubular epithelial cells. Conclusion: The C5a of the complement C5 can be combined with the C5aR on the renal tubular epithelial cells. To regulate the production of IL-17 in renal tubular epithelial cells and to promote the occurrence of allograft rejection, the results of this study can be complement inhibition.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R699.2

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