本文關(guān)鍵詞：調(diào)節(jié)性B細(xì)胞在銀屑病發(fā)病中的作用研究，由筆耕文化傳播整理發(fā)布。

        在免疫學(xué)研究領(lǐng)域中免疫調(diào)節(jié)研究占據(jù)了其中重要的部分，調(diào)節(jié)性細(xì)胞對(duì)于免疫平衡和自身穩(wěn)定的維持具有重要作用。傳統(tǒng)上人們認(rèn)為B細(xì)胞在免疫系統(tǒng)中發(fā)揮正向免疫調(diào)節(jié)作用，然而最近研究發(fā)現(xiàn)B細(xì)胞也具有負(fù)向免疫調(diào)節(jié)功能。1996年，Janeway等首次在實(shí)驗(yàn)性自身免疫性腦脊髓炎（EAE）中發(fā)現(xiàn)了具有抑制功能的B細(xì)胞的存在，1997年Bhan等在慢性結(jié)腸炎中發(fā)現(xiàn)B細(xì)胞能抑制T細(xì)胞介導(dǎo)的炎癥反應(yīng)，并首次提出“調(diào)節(jié)性B細(xì)胞”的概念。隨后又有研究者在多種自身免疫性疾病和炎癥性疾病的小鼠模型中及在變態(tài)反應(yīng)、腫瘤免疫、抗感染免疫中都證實(shí)B細(xì)胞具有負(fù)向免疫調(diào)節(jié)功能。2008年Tedder研究組首次在小鼠脾臟中鑒定出發(fā)揮負(fù)向免疫調(diào)節(jié)功能的B細(xì)胞亞群，其表型為CD19+CD5+CD1dhi，主要通過(guò)分泌IL-10來(lái)發(fā)揮負(fù)向免疫調(diào)節(jié)作用，并將其稱為B10細(xì)胞。通過(guò)小鼠實(shí)驗(yàn)發(fā)現(xiàn)B10細(xì)胞的存在和功能后，人們進(jìn)一步研究發(fā)現(xiàn)人類體內(nèi)也存在B10細(xì)胞。2009年Blair等在系統(tǒng)性紅斑狼瘡患者體內(nèi)發(fā)現(xiàn)了調(diào)節(jié)性B細(xì)胞亞群的存在，其表型為CD19+CD24hiCD38hi，并發(fā)現(xiàn)同小鼠調(diào)節(jié)性B細(xì)胞一樣其負(fù)向免疫調(diào)節(jié)功能依賴于IL-10。2010年Yohei Iwata等在自身免疫性疾病包括系統(tǒng)性紅斑狼瘡、風(fēng)濕性關(guān)節(jié)炎、干燥綜合征、自身免疫性皰病及多發(fā)性硬化中發(fā)現(xiàn)調(diào)節(jié)性B細(xì)胞的存在，其負(fù)向免疫調(diào)節(jié)功能的發(fā)揮依賴于IL-10。銀屑病是在一定的遺傳背景與環(huán)境因素相互作用下由Th1/Th17異�；罨閷�(dǎo)的自身免疫性疾病，負(fù)向免疫調(diào)節(jié)功能障礙在其發(fā)病過(guò)程中發(fā)揮重要作用，但其確切的細(xì)胞和分子機(jī)制尚不明確。銀屑病發(fā)病過(guò)程不僅存在CD4+CD25+Treg數(shù)量和功能異常而且伴有IL-10水平的變化。小鼠研究發(fā)現(xiàn)B細(xì)胞能夠影響Treg的發(fā)育和功能，調(diào)節(jié)性B細(xì)胞的部分負(fù)向免疫調(diào)節(jié)功能是通過(guò)CD4+CD25+Treg實(shí)現(xiàn)的。這些均提示著調(diào)節(jié)性B細(xì)胞可能參與了銀屑病的發(fā)病并發(fā)揮著重要的負(fù)向調(diào)控作用。本研究比較分析了銀屑病患者外周血調(diào)節(jié)性B細(xì)胞的數(shù)量、表型及功能，對(duì)調(diào)節(jié)性B細(xì)胞在銀屑病發(fā)病中的可能作用進(jìn)行了初步的分析。主要研究?jī)?nèi)容和結(jié)果如下：1.銀屑病患者外周血B10細(xì)胞分析：收集銀屑病患者外周血，分離PBMC，給予CpG+CD40L刺激，并加入PIB（P為PMA，I為離子霉素，B為BrefeldinASolution，前兩者刺激細(xì)胞因子合成，后者抑制細(xì)胞因子分泌），進(jìn)行5h培養(yǎng)，收集細(xì)胞，標(biāo)記CD19及胞內(nèi)IL-10，以流式細(xì)胞儀檢測(cè)分析。結(jié)果發(fā)現(xiàn)：銀屑病患者外周血IL-10+B細(xì)胞比例均數(shù)降低，但整體與正常對(duì)照間無(wú)統(tǒng)計(jì)學(xué)差異。2.銀屑病患者外周血B10前體細(xì)胞分析：收集患者外周血，分離PBMC，加入CpG+CD40L刺激，并加入PIB，進(jìn)行48h培養(yǎng)，收集細(xì)胞，標(biāo)記CD19及胞內(nèi)IL-10，以流式細(xì)胞儀檢測(cè)分析。結(jié)果發(fā)現(xiàn)：銀屑病患者外周血IL-10+B細(xì)胞比例與正常對(duì)照相比明顯升高，表明B10前體細(xì)胞數(shù)量增多。3.銀屑病患者外周血B10細(xì)胞分化能力分析：對(duì)比分析銀屑病患者與正常人外周血在不同刺激條件下培養(yǎng)48小時(shí)后的B10細(xì)胞比例，結(jié)果發(fā)現(xiàn)銀屑病患者外周血在體外刺激培養(yǎng)條件下產(chǎn)生的B10細(xì)胞占B細(xì)胞總數(shù)的比例與正常人比較沒有差異，表明B10細(xì)胞體外刺激條件下不存在分化障礙。4.治療后銀屑病患者外周血B10前體細(xì)胞比例分析：收集同一患者治療前后外周血，分離PBMC，同上四組刺激后收集細(xì)胞標(biāo)記CD19及胞內(nèi)IL-10，以流式細(xì)胞儀檢測(cè)分析。結(jié)果發(fā)現(xiàn)治療后銀屑病患者外周血B10前體細(xì)胞數(shù)明顯低于治療前。5.銀屑病患者B10細(xì)胞表型分析：收集銀屑病患者及正常人外周血，分離PBMC，同上四組刺激后收集細(xì)胞標(biāo)記CD19、CD38、CD24。以流式細(xì)胞儀檢測(cè)分析，，結(jié)果發(fā)現(xiàn)在PIB基礎(chǔ)刺激或者CD40L及CpG單獨(dú)刺激時(shí)分泌IL10的B細(xì)胞主要位于CD24hiCD38low或CD24hiCD38-區(qū)域，而在PIB+CD40L+CpG時(shí)B細(xì)胞所分泌IL10增加且主要位于CD24hiCD38hi區(qū)域。6.銀屑病患者外周血B10細(xì)胞功能分析：收集銀屑病患者及正常人外周血，分離PBMC，表面標(biāo)記CD19、CD38、CD24。流式細(xì)胞儀分選CD19+CD24hiCD38hiB細(xì)胞及除外CD19+CD24hiCD38hiB細(xì)胞的其他細(xì)胞。將含有CD19+CD24hiCD38hiB細(xì)胞和不含有CD19+CD24hiCD38hiB細(xì)胞的PBMC分別置于預(yù)先包被抗CD3抗體的24孔板培養(yǎng)72h，于最后6h加入P+I刺激，收集上清，ELISA檢測(cè)IFN–γ和TNF-α。結(jié)果發(fā)現(xiàn)正常人CD19+CD24hiCD38hiB10細(xì)胞可以明顯抑制T細(xì)胞分泌TNF-α，而患者B10細(xì)胞的抑制作用不明顯。結(jié)論：本研究首次揭示了銀屑病患者外周血B10細(xì)胞數(shù)目和功能異常，高度提示B10細(xì)胞在銀屑病發(fā)病中發(fā)揮負(fù)向調(diào)控作用，其確切的作用和機(jī)制尚有待進(jìn)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.

          調(diào)節(jié)性B細(xì)胞在銀屑病發(fā)病中的作用研究

縮略語(yǔ)表5-8中文摘要8-11Abstract11-14前言15-17文獻(xiàn)回顧17-34    1.調(diào)節(jié)性B細(xì)胞的特性17-22    2.B10細(xì)胞負(fù)向免疫調(diào)節(jié)功能22-26    3.人B10細(xì)胞26-30    4.B10細(xì)胞與銀屑病30-34材料34-37方法37-41結(jié)果41-49討論49-53小結(jié)53-54參考文獻(xiàn)54-68個(gè)人簡(jiǎn)歷和研究成果68-69致謝69-70

  本文關(guān)鍵詞：調(diào)節(jié)性B細(xì)胞在銀屑病發(fā)病中的作用研究，由筆耕文化傳播整理發(fā)布。