调节性B细胞在银屑病发病中的作用研究

  本文关键词：调节性B细胞在银屑病发病中的作用研究，由笔耕文化传播整理发布。

        在免疫学研究领域中免疫调节研究占据了其中重要的部分，调节性细胞对于免疫平衡和自身稳定的维持具有重要作用。传统上人们认为B细胞在免疫系统中发挥正向免疫调节作用，然而最近研究发现B细胞也具有负向免疫调节功能。1996年，Janeway等首次在实验性自身免疫性脑脊髓炎（EAE）中发现了具有抑制功能的B细胞的存在，1997年Bhan等在慢性结肠炎中发现B细胞能抑制T细胞介导的炎症反应，并首次提出“调节性B细胞”的概念。随后又有研究者在多种自身免疫性疾病和炎症性疾病的小鼠模型中及在变态反应、肿瘤免疫、抗感染免疫中都证实B细胞具有负向免疫调节功能。2008年Tedder研究组首次在小鼠脾脏中鉴定出发挥负向免疫调节功能的B细胞亚群，其表型为CD19+CD5+CD1dhi，主要通过分泌IL-10来发挥负向免疫调节作用，并将其称为B10细胞。通过小鼠实验发现B10细胞的存在和功能后，人们进一步研究发现人类体内也存在B10细胞。2009年Blair等在系统性红斑狼疮患者体内发现了调节性B细胞亚群的存在，其表型为CD19+CD24hiCD38hi，并发现同小鼠调节性B细胞一样其负向免疫调节功能依赖于IL-10。2010年Yohei Iwata等在自身免疫性疾病包括系统性红斑狼疮、风湿性关节炎、干燥综合征、自身免疫性疱病及多发性硬化中发现调节性B细胞的存在，其负向免疫调节功能的发挥依赖于IL-10。银屑病是在一定的遗传背景与环境因素相互作用下由Th1/Th17异常活化介导的自身免疫性疾病，负向免疫调节功能障碍在其发病过程中发挥重要作用，但其确切的细胞和分子机制尚不明确。银屑病发病过程不仅存在CD4+CD25+Treg数量和功能异常而且伴有IL-10水平的变化。小鼠研究发现B细胞能够影响Treg的发育和功能，调节性B细胞的部分负向免疫调节功能是通过CD4+CD25+Treg实现的。这些均提示着调节性B细胞可能参与了银屑病的发病并发挥着重要的负向调控作用。本研究比较分析了银屑病患者外周血调节性B细胞的数量、表型及功能，对调节性B细胞在银屑病发病中的可能作用进行了初步的分析。主要研究内容和结果如下：1.银屑病患者外周血B10细胞分析：收集银屑病患者外周血，分离PBMC，给予CpG+CD40L刺激，并加入PIB（P为PMA，I为离子霉素，B为BrefeldinASolution，前两者刺激细胞因子合成，后者抑制细胞因子分泌），进行5h培养，收集细胞，标记CD19及胞内IL-10，以流式细胞仪检测分析。结果发现：银屑病患者外周血IL-10+B细胞比例均数降低，但整体与正常对照间无统计学差异。2.银屑病患者外周血B10前体细胞分析：收集患者外周血，分离PBMC，加入CpG+CD40L刺激，并加入PIB，进行48h培养，收集细胞，标记CD19及胞内IL-10，以流式细胞仪检测分析。结果发现：银屑病患者外周血IL-10+B细胞比例与正常对照相比明显升高，表明B10前体细胞数量增多。3.银屑病患者外周血B10细胞分化能力分析：对比分析银屑病患者与正常人外周血在不同刺激条件下培养48小时后的B10细胞比例，结果发现银屑病患者外周血在体外刺激培养条件下产生的B10细胞占B细胞总数的比例与正常人比较没有差异，表明B10细胞体外刺激条件下不存在分化障碍。4.治疗后银屑病患者外周血B10前体细胞比例分析：收集同一患者治疗前后外周血，分离PBMC，同上四组刺激后收集细胞标记CD19及胞内IL-10，以流式细胞仪检测分析。结果发现治疗后银屑病患者外周血B10前体细胞数明显低于治疗前。5.银屑病患者B10细胞表型分析：收集银屑病患者及正常人外周血，分离PBMC，同上四组刺激后收集细胞标记CD19、CD38、CD24。以流式细胞仪检测分析，，结果发现在PIB基础刺激或者CD40L及CpG单独刺激时分泌IL10的B细胞主要位于CD24hiCD38low或CD24hiCD38-区域，而在PIB+CD40L+CpG时B细胞所分泌IL10增加且主要位于CD24hiCD38hi区域。6.银屑病患者外周血B10细胞功能分析：收集银屑病患者及正常人外周血，分离PBMC，表面标记CD19、CD38、CD24。流式细胞仪分选CD19+CD24hiCD38hiB细胞及除外CD19+CD24hiCD38hiB细胞的其他细胞。将含有CD19+CD24hiCD38hiB细胞和不含有CD19+CD24hiCD38hiB细胞的PBMC分别置于预先包被抗CD3抗体的24孔板培养72h，于最后6h加入P+I刺激，收集上清，ELISA检测IFN–γ和TNF-α。结果发现正常人CD19+CD24hiCD38hiB10细胞可以明显抑制T细胞分泌TNF-α，而患者B10细胞的抑制作用不明显。结论：本研究首次揭示了银屑病患者外周血B10细胞数目和功能异常，高度提示B10细胞在银屑病发病中发挥负向调控作用，其确切的作用和机制尚有待进一步研究。

    The research on immune regulation is of great importance in field ofimmunology. Regulatory B cells （Breg） play a very important role in immunebalance and homeostasis. Traditionally, people thought that B cells had the abilityof positive immune modulation in the immune system. However, accumulatingdata showed that B cells also had the ability of negative immune modulation. In1996, Janeway group firstly found that B cells had the inhibitory anilities inexperimental autoimmune encephalomyelitis （EAE）. In1997, for the first time,Bhan et al proposed the conception of―Regulatory B cells‖, which was resultedfrom the fact that B cells could inhibit the inflammation Induced by T cells inchronic colitis. Thereafter, researchers demonstrated that B cells have thefunction of negative immune modulation in the mouse models of severalautoimmunity diseases, inflammation diseases, allergy, tumor immunity andanti-infection immunity. In2008, Tedder group identified that the sub-populationof B cells had the ability of negative immune modulation by producing IL-10,was and those cells were called as B10cells and the phenotypes wereCD19+CD5+CD1dhi.Studies also showed that there are B10cells in human.In2009, Blair et aldemonstrated that regulatory B cells existed in systemic lupus erythematosus （SLE） patients, and the phenotype was CD19+CD24hiCD38hi. The negativeimmune modulation of regulatory B cells in human is also dependent on theproduction of IL-10, which is the same as Breg in mice. In2010, Yohei Iwata etal got the similar discovery that regulatory B cells existed in patient ofautoimmune diseases, such as SLE, rheumatoid arthritis, primary Sj gren’ssyndrome, autoimmune vesiculobullous skin disease and multiple sclerosis. Theinhibitory immune modulation was dependent on the production of IL-10.The compelling evidence showed that psoriasis is a Th1/Th17cellsdominant autoimmune disease, and there are aberrant regulatory function inpsoriasis patient. The abnormality of the quantity and function CD4+CD25+Tregand the variation of IL-10expression have been observed in the pathogenesis ofpsoriasis. In mice, it is has been demonstrated that B cells could have effect onthe development and function of Treg. The negative immune modulation ofregulatory B cells is partly dependent on CD4+CD25+Treg. Based on the abovestudies, we hypothesized that the abnormality of the quantity and functionCD4+CD25+Treg might be induced by regulatory B cells in psoriasis patients.We investigated the number, phenotype, function of B10cells in psoriasis. Theresults are as the following:1. The analysis on the number of B10cells in psoriasis patients: Bloodsamples were collected, and PBMCs were separated for culture. CpG+CD40Land PIB （PMA, ionomycin and Brefeldin A Solution） were added for5h culturing,then the cells were collected and stained with CD19and IL-10and subjectedfor FCM analysis. We found that the number of IL-10+B cells in psoriaticperipheral blood decreased compared with the healthy control group.2. The analysis of number of B10-pro cells in psoriasis patients: Bloodsamples were collected, and PBMCs were separated for culture. CpG+CD40L and PIB （PMA, ionomycin and Brefeldin A Solution） were added for48hculturing, then the cells were collected and stained with CD19and IL-10andsubjected for FCM analysis. We found that the numberof IL-10+B cells inpsoriatic peripheral blood elevateded compared with the healthy control group,which means that the number of B10-pro increased.3. The analysis of differentiation abilities of B10cells in psoriatic patient.Under different groups of stimuli the ratios of pro-B10cells were detectedbetween psoriasis patients and healthy controls. We found that there was nodifference in the differentiation abilities of B10cells between psoriasis patientsand normal control.4. The analysis of number of B10-pro cells in psoriatic peripheral blood afterthe treatments. The PBMC were treated as describled above. We found that afterthe treatments number of B10-pro cells were less than that before the treatmentsand healthy controls.5. The analysis on the phenotype of B10cells in psoriasis patients: Bloodsamples were collected, and PBMCs were separated and subjected for CD19,CD38and CD24staining after culture with four groups of stimulis. Then the cellswere analyzed by FCM. We found that under PIB or CD40L and CpGstimulating, the IL-10-secreting B cells showed mainly CD24hiCD38loworCD24hiCD38-phenotype, and under the PIB+CD40L+CpG stimulating, the ratioof IL-10secreting cells increased and mainly with CD24hiCD38hiphenotype.6. The analysis on the function of B10cells in psoriatic patient. Bloodsamples were collected, and PBMCs were separated and subjected for CD19,CD38and CD24staining. Then the CD19+CD24hiCD38hiB cells were sorted byFCM. Meanwhile, other cells excluded CD19+CD24hiCD38hiB cells were alsocollected. We compared the culture with and without CD19+CD24hiCD38hiB cells in the24wells plate-coated with anti-CD3for72h. We stimulated thesecells with P+I for last6h and collected the supernatant, and IFN-γ and TNF-αwere detected by ELISA. We found that the healthy persons’ B10significantlyinhibited the lymphocytes’ production of TNF-α, but the psoriasis patients’ B10couldn’t.Conclusion: Our studies showed for the first time that the number andfunction of B10cells were abnormal in psoriasis patient,indicating that B10cellsmight play a negative regulatory role in the progress of psoriasia. More studiesare needed to further our understanding of the role and mechanism of B10cells inpsoriasis.

          调节性B细胞在银屑病发病中的作用研究

缩略语表5-8中文摘要8-11Abstract11-14前言15-17文献回顾17-34    1.调节性B细胞的特性17-22    2.B10细胞负向免疫调节功能22-26    3.人B10细胞26-30    4.B10细胞与银屑病30-34材料34-37方法37-41结果41-49讨论49-53小结53-54参考文献54-68个人简历和研究成果68-69致谢69-70

  本文关键词：调节性B细胞在银屑病发病中的作用研究，由笔耕文化传播整理发布。