解毒活血法抗大鼠肾移植急性排斥机理研究

Acute renal allograft rejection is the leading cause of late renal transplant failure. Many immune and non-immune factors contribute to the acute rejection that lead to chronic renal failure and it is hard to prevent kidney failure at early stage after transplantation. The effect of Chinese herb of clearing away toxic material and activating blood circulation method on prevention of acute renal allograft rejection has not been determined. The aim of the study is to evaluate the therapeutic effect of clearing away toxic material and activating blood circulation method，using Salvea Miltiorrhiza and Oldenlandia Diffusa on experimental acute allograft rejection model with microsurgery. BN to LEW, LEW to LEW renal transplantation will be performed and recipients will be treated with Salvea Miltiorrhiza and Oldenlandia Diffusa. The serum creatinine levels, proteinuria, renal functional,survival time of recipients and pathological changes, according to the Banff scheme, will be observed at 2th, 4th, 7th days posttransplantation. Kidney graft will be harvested at 2th, 4th, and 7th days. The level of IL-2、IFN-γ、IL-6、IL-10 will be detected by ELISA. The expressions of ED-1, CD4+, CD8+, MHC-Ⅱ, TLR4mRNA、miR-210, will be analyzed by quantitative reverse-transcriptase polymerase chain reaction and immunohistochemistry. And compared with the untreated, CSA treated, allograft controls. This result will suggest that the Chinese herb of clearing away toxic material and activating blood circulation method can prevent acute renal allograft rejection. The research might offer some new approaches to inhibiting the acute renal allograft rejection and extend the life of transplanted kidney.

急性排异反应（Acute Rejection，AR）是临床上最常见的排异反应，可发生在移植后的任何时间内。早期AR与晚期AR均为移植物长期存活的独立危险因素，而晚期AR危害更大，目前还未找到完全克服或不发生AR的方法。本研究围绕解毒活血抗肾移植AR这一基本问题，探讨蛇舌草与丹参对大鼠肾移植AR影响及其免疫学及非免疫学作用机理，将填补解毒活血中药抗肾移植AR的实验研究空白。运用显微外科手术方法制作大鼠肾移植AR经典模型，观察模型大鼠血常规、肝肾功能、血脂、24小时尿蛋白定量、肌酐清除率，生存时间,并对肾组织进行病理学检查，运用ELISA法检测大鼠血清中IL-2、 IFN-γ、IL-6、IL-10含量,运用免疫组化与分子生物学方法探讨ED-1、CD4+、CD8+、MHC-Ⅱ、TLR4mRNA、miR-210在肾移植AR大鼠肾组织中的表达及蛇舌草和丹参对移植肾病理的影响。

急性排斥反应是困扰器官移植学的主要难题之一，中药抗移植物排斥的问题在国内外已引起注意。近年来，在抗排斥反应中药研究中清热解毒中药越来越受到重视。 通过BN和Lewis大鼠为供、受体，建立大鼠肾移植急性排斥模型，将模型大鼠随机分为对照组、丹参组、白花蛇草、丹参合白花蛇草组、环孢素（CSA）组、CSA合丹参白花蛇草组，并进行用药观察。观察移植后受体大鼠存活时间、受体大鼠第6天24小时尿蛋白定量、肾功能、移植肾形态学变化，观察免疫组化染色肾组织中ED-1、CD4+、CD8+ 、MHC-Ⅱ阳性细胞表达，用RT-PCR方法检测肾皮质TLR4mRNA及miR-210表达，ELISA法检测大鼠血清中IL-2、 IFN-γ、IL-6、IL-10含量。研究表明活血解毒法之丹参及白花蛇草具有延长移植肾存活、改善移植肾功能减退、减轻移植肾组织病理损害、降低肾移植急性排斥血清IL-2、IFN-γ含量、降低肾移植肾组织中质TLR4mRNA、miR-210相对表达。提示活血解毒法对大鼠肾移植急性排斥的发病过程中细胞及体液免疫反应起着一定作用。研究结果展示了应用活血解毒法之丹参及白花蛇草防治急性排斥具有一定移植肾保护作用，与CSA具有协同抗急性排斥作用。为解毒活血中药抗肾移植急性排斥提供了实验依据。