【作者】 程悦；

【导师】 袁发焕；

【作者基本信息】 第三军医大学 ， 内科学， 2009， 博士

【摘要】 研究背景在肾脏疾病如肾小球肾炎、小管间质性肾炎及同种异体移植物排斥反应的炎症反应中,T细胞在对肾脏免疫性损伤中占主导作用。T细胞的活化不仅需要MHC-肽-TCR方式提供第一信号,还需要共刺激分子提供第二信号。如果没有足够的共刺激信号刺激,T细胞表现为失能、耐受和凋亡。程序性死亡1共刺激分子(programmed death gene 1,PD-1)/PD-L(PD-1 ligand)途径是新近发现的共刺激信号,属CD28/B7超家族成员。已证实该信号途径的异常表达参与了自身免疫性疾病、同种异体移植物排斥反应及各种感染的发生及进展。目前针对共刺激信号途径的调节已经成为免疫治疗的新方法。T细胞对疾病的影响不仅体现在活化水平,还体现在选择性分化水平。T-bet是新发现的Th1特异性转录因子,可促进Th0细胞向Th1细胞分化,GATA-3是Th2特异的转录因子,在Th2细胞分化过程中起关键作用。当机体免疫功能异常时,Th0细胞分化的Th1和Th2细胞亚群比例及功能失衡,出现相应的Th1和Th2漂移性疾病。研究显示T-bet/GATA-3比值能较准确地反映Th1/Th2细胞亚群的平衡状态,可用于评价慢性肾炎的免疫平衡状态。通过调节T-bet/GATA-3而恢复Th1/Th2的平衡,被认为是自身免疫性疾病的一个可能的治疗方向。盐酸青藤碱(sinomenine, SN)是从中药青风藤(Sinomenium scutum Rehd et wils)中提取的生物碱单体,为新型中药免疫调节药物,疗效较好副作用小,用于慢性肾炎的治疗已有10多年的成功经验,但其具体机制依然不清。本研究以常见的非IgA系膜增生性肾炎(mesangial proliferative nephritis,MsPGN)为观察对象(避免不同病理类型对结果的影响),观察这类患者体内PD-1/PD-L表达以及Th1/Th2平衡状态有何变化;并从临床的角度,前瞻性地观察SN对MsPGN患者蛋白尿等临床症状的影响,观察SN对患者外周血单个核细胞(peripheral blood mononuclear cell,PBMC) PD-1/PD-L、T-bet/GATA-3及Th1/Th2细胞因子表达的影响,以探讨SN治疗MsPGN的免疫机制,为应用SN治疗MsPGN提供理论依据。研究方法1.研究对象实验观察MsPGN患者25例,另观察10例正常人作为对照。其中肾组织标本,MsPGN组标本来自25例MsPGN患者的肾活检标本,对照组标本10例来自亲属供肾的移植前肾活检标本和因肾肿瘤而切除的远离肿瘤端且经光镜证实正常的肾组织标本。2.实验方法(1)免疫组化检测治疗前MsPGN患者肾组织和对照组肾组织中PD-1、PD-L1、PD-L2蛋白的表达;定量RT-PCR和多色流式细胞技术检测治疗前MsPGN患者和对照组PBMC PD-1、PD-L1、PD-L2 mRNA和蛋白表达。以了解MsPGN患者是否存在共刺激信号PD-1/PD-L的异常表达。(2)免疫组化检测治疗前MsPGN患者肾组织和对照组肾组织中T-bet、GATA-3蛋白的表达;定量RT-PCR检测治疗前MsPGN患者和对照组PBMC T-bet、GATA-3 mRNA表达;同时采用ELISA法测定治疗前MsPGN患者和对照组血清IFN-γ、IL-4、IL-10表达水平。以了解MsPGN患者在T细胞选择性分化水平是否存在Th1/Th2失衡。(3)采用定量RT-PCR和多色流式细胞技术观察不同浓度SN(50μg/ml-200μg/ml)对体外培养的MsPGN患者外周血淋巴细胞PD-1、PD-L1、PD-L2 mRNA和蛋白表达的影响。(4)采用定量RT-PCR和Western Blot方法观察不同浓度SN(50μg/ml-200μg/ml)对体外培养的MsPGN患者外周血淋巴细胞T-bet、GATA-3 mRNA和蛋白表达的影响;同时采用ELISA法检测培养上清中IFN-γ、IL-4、IL-10表达变化。(5)留取对照组和治疗前后MsPGN患者血、尿标本,分别进行临床生化及尿蛋白检测,以观察SN治疗MsPGN的临床疗效。(6)采用定量RT-PCR和多色流式细胞技术观察MsPGN患者接受SN治疗1月、3月后PBMC PD-1、PD-L1、PD-L2 mRNA和蛋白表达的变化。(7)采用定量RT-PCR观察MsPGN患者接受SN治疗1月、3月后PBMC T-bet、GATA-3 mRNA表达的变化;同时采用ELISA法检测血清IFN-γ、IL-4、IL-10表达变化。结果1. MsPGN患者肾活检标本肾小管间质部位的淋巴细胞有PD-1阳性表达,而PD-L1表达在肾小管上皮细胞(renal tubular epithelial cells, TEC),肾小球无PD-1、PD-L1表达。对照组肾组织中无PD-1表达,PD-L1表达很少甚至无表达。两组肾组织标本中均未检测到PD-L2表达。MsPGN患者PBMC PD-1、PD-L1 mRNA和蛋白表达高于对照组,而PD-L2蛋白在MsPGN患者和对照组中表达很少,两组间无显著差异。2.在TEC和肾间质渗出的淋巴细胞检测到T-bet、GATA-3的阳性表达,MsPGN肾组织T-bet表达强于对照组而GATA-3表达弱于对照组;MsPGN患者PBMC T-bet mRNA表达高于对照组,而GATA-3 mRNA表达低于对照组;MsPGN患者血清IFN-γ水平高于对照人群,而IL-10水平低于对照人群。3.体外实验观察到SN可以抑制MsPGN患者外周血淋巴细胞PD-1、PD-L1 mRNA和蛋白表达,这种抑制作用随着SN浓度的增加而增强。4.体外实验观察到SN可以抑制MsPGN患者外周血淋巴细胞T-bet mRNA和蛋白表达,下调T-bet/GATA-3比值,并抑制Th1细胞因子IFN-γ的表达,这种调节作用随着SN浓度的增加而增强。5. SN治疗可有效控制MsPGN患者蛋白尿,并升高补体C3水平,副作用小。6. SN治疗可以抑制MsPGN患者PBMC PD-1、PD-L1 mRNA和蛋白表达,这种抑制作用随着SN治疗时间的延长而增强。7. SN治疗可以抑制MsPGN患者PBMC T-bet的mRNA表达,下调T-bet/GATA-3比值,并抑制Th1细胞因子IFN-γ的表达,这种调节作用随着SN治疗时间的延长而增强。结论SN治疗可有效控制轻、中度MsPGN患者蛋白尿,并升高补体C3水平,副作用小,但疗程至少要1月以上才有明显疗效。MsPGN患者肾组织和PBMC上PD-1、PD-L1表达高于对照组,可能与MsPGN的免疫发病机制有关。SN可以抑制PD-1、PD-L1的病理性高表达,这种抑制作用随着SN浓度的增加、治疗时间的延长而增强,提示SN控制MsPGN患者蛋白尿的治疗机制可能与抑制PD-1、PD-L1表达有关。MsPGN患者在T细胞选择性分化过程中,在转录调控水平和细胞因子水平存在Th1优势反应,这可能是MsPGN的重要免疫发病机制。而SN能抑制MsPGN患者T-bet表达,下调T-bet/GATA-3比值,并抑制Th1细胞因子IFN-γ的表达,从而在转录调控水平和细胞因子水平抑制Th1反应,恢复Th1/Th2的平衡。SN的这种调节作用随着SN浓度的增加、治疗时间的延长而增强。提示SN恢复MsPGN患者Th1/Th2平衡的免疫调节作用,可能是其能控制蛋白尿的重要的治疗机制。更多还原

【Abstract】 BackgroundExperimental results from inflammatory renal diseases, such as allograft rejection, tubulointerstitial nephritis and glomerulonephritis, have suggested a prominent role of T cells in the injurious renal immune response. It is accepted that signals provide by TCR-peptide-MHC and co-stimulatory molecules are required for the optimal activation of T cells. Without sufficient co-stimulation, T cells are rendered anergy, tolerance or apoptosis. Recently, B7 superfamily molecules, PD-L1, PD-L2 and their receptor PD-1 (programmed death-1) have been identified. Previous studies indicated that the abnormality of PD-1/PD-L pathway might play a role in the development of autoimmune disease, allograft rejection, and various kinds of infection. Treatment aiming at regulating co-stimulatory pathway has now been recognized as a new way of immunotherapy.Inappropriate T cell responses result from not only the abnormality of T cell activation but also the abnormality of T cell differentiation. Immune responses polarized by either Th1 or Th2 subset predominance result in the activation of different inflammatory effector pathways and disease outcomes. T-bet is a newly discovered Th1-specific transcription factor thought to initiate Th1 development, whereas GATA-3 plays a pivotal role in the development of the Th2 phenotype. It has been reported that the T-bet/GATA-3 ratio is representative of the balance between Th1 and Th2 cells. Maintaining the Th1/Th2 balance via the regulation of T-bet/GATA-3 ratio has been recognized as a promising therapy in the treatment of autoimmune diseases.Sinomenine (SN), a pure alkaloid extracted from the Chinese medical plant Sinomenium acutum, has been effectively used for over ten years in the treatment of patients with chronic glomerulonephritis. Although sinomenine is effectively used in the clinic and is gaining international popularity, little is known about its exact mechanisms of action.In this study, patients with mesangial proliferative nephritis (MsPGN), which is one of the most common pathological type of chronic GN, were examined. Both the PD-1/PD-L expression and the balanced state of Th1/Th2 were observed in patients with MsPGN. Then the effect of SN on proteinuria was detected. In order to elucidate the therapeutic mechanism of SN, the effect of SN on the expressions of PD-1/PD-L and T-bet/GATA-3 in peripheral blood mononuclear cells (PBMCs) and the serum levels of interferon-γ(IFN-γ), interleukin (IL)-4, and IL-10 were studied.Patients and methods1. Patients and renal specimensA total of 25 patients with MsPGN and 10 healthy donors were enrolled in this study. Immunohistochemistry was enforced on the paraffin-embedded tissues from renal biopsies of 25 patients with MsPGN. Additionally, ten specimens obtained from pre-transplant renal biopsies and non-tumor kidney tissues from cancer patients served as controls.2. methods(1) In order to observe the expression pattern of PD-1/PD-L in patients with MsPGN, the intrarenal levels of PD-1, PD-L1, and PD-L2 were determined by immunohistochemistry and the expression levels of PD-1, PD-L1, and PD-L2 on PBMCs in both MsPGN patients and healthy donors were detected via real-time RT-PCR and flow cytometry.(2) In order to observe if there is an imbalance between Th1 and Th2 cells in patients with MsPGN, the intrarenal levels of T-bet and GATA-3 were determined by immunohistochemistry, the expression levels of T-bet and GATA-3 in PBMCs in both MsPGN patients and healthy donors were detected via real-time RT-PCR, and the serum levels of IFN-γ, IL-4, and IL-10 were studied via ELISA.(3) The effect of SN on the expression levels of PD-1, PD-L1, and PD-L2 on lymphocytes in vitro was studied via real-time RT-PCR and flow cytometry.(4) The effect of SN on the expression levels of T-bet and GATA-3 on lymphocytes in vitro was studied via real-time RT-PCR and Western Blot. Additionally, ELISA was applied to detect the effect of SN on the supernatant levels of IFN-γ, IL-4, and IL-10.(5) To evaluate the clinic effect of SN, blood biochemical parameters and proteinuria were detected at month 0, month 1, and month 3 in the course of treatment with SN.(6) The effect of SN on the expression levels of PD-1, PD-L1, and PD-L2 on PBMCs in vivo was studied via real-time RT-PCR and flow cytometry at 1 month and 3 months after the start of treatment with SN.(7) The effect of SN on the expression levels of T-bet and GATA-3 on PBMCs in vivo was studied via real-time RT-PCR and the serum levels of IFN-γ, IL-4, and IL-10 were studied by ELISA at 1 month and 3 months after the start of treatment with SN.Results1. Positive staining for PD-1 was detected on infiltrating lymphocytes in the interstitial space and PD-L1 was detected in tubular epithelial cells in renal tissues from MsPGN patients. However, the expression of PD-L2 was hardly observed in renal tissues in both MsPGN patients and control. Patients with MsPGN were found to have increased PD-1 and PD-L1 expression in both renal biopsy tissues and PBMCs compared with normal individuals.2. Positive staining for both T-bet and GATA-3 was detected in tubular epithelial cells and infiltrating lymphocytes in the interstitial space. Patients with MsPGN were found to have increased T-bet and depressed GATA-3 expression in both renal biopsy tissues and PBMCs compared with normal individuals. Additionally, MsPGN patients were found to have elevated serum IFN-γvalues and decreased serum IL-10 values when compared with healthy donors.3. The expression of PD-1 and PD-L1 on lymphocytes was suppressed by SN in vitro, and the inhibition effect was concentration dependent.4. The expression of T-bet on lymphocytes was suppressed by SN in vitro, therefore, the T-bet/GATA-3 ratio was markedly decreased. SN was also found to markedly inhibit the secretion of IFN-γ. These effects above were concentration dependent.5. SN could ameliorate proteinuria and increase the levels of Complement C3 in MsPGN patients with rare side effects.6. SN could suppresse the expression of PD-1 and PD-L1 on PBMCs from MsPGN patients in vivo, and this effect was more significant at 3 months.7. SN was found to cause a decrease in T-bet expression on PBMCs from MsPGN patients in vivo, resulting in a drop in the T-bet/GATA-3 ratio. SN could also inhibit the secretion of IFN-γ. These effects were more significant at 3 months.ConclusionsSN could ameliorate proteinuria and increase the levels of Complement C3 in MsPGN patients with rare side effects, however, the course of treatment should last for more than 1 month.Increased expression of PD-1 and PD-L1 in renal tissues and PBMCs from MsPGN patients may contribute to the development of MsPGN. Our results indicate that SN has the potential to inhibit the high expression of PD-1 and PD-L1 in PBMCs from MsPGN patients and thereby produce therapeutic effects.A shift toward the Th1 pathway of Th cell activation at both transcriptional level and cytokine level occurs in MsPGN patients, and that SN has the potential to counter this shift in the Th1/Th2 balance and thereby produce therapeutic effects.更多还原