【作者】 姜廷帅；

【导师】 王雨生； 蔡莉；

【作者基本信息】 第四军医大学 ， 眼科学， 2010， 博士

【摘要】 研究背景:角膜移植手术是治疗不可逆角膜盲的最重要手段,我国每年大约有3000例各种角膜疾病患者需要接受角膜移植手术。由于角膜赦免状态的存在,在所有固体器官移植中,角膜移植是成功率最高的手术。即便如此,角膜移植术后植片的长期存活率并不令人满意,排斥反应的发生仍然无法避免。长期随访观察结果表明,对于低危受体,移植后1年成功率高达90%,但10年后下降为60%～70%;而对于眼部有新生血管、严重化学烧伤多次移植失败等高危病例,10年后角膜移植成功率小于35%[107]。因此,进一步深入了解角膜移植排斥反应的发生机制及其预防、治疗方法,仍是眼科免疫领域的重要课题。研究表明,调节性T细胞(Treg)广泛存在于小鼠、大鼠、人的中枢和周围免疫系统中,在维持体内的免疫平衡,抑制器官移植排斥反应、自身免疫性疾病以及控制肿瘤的免疫治疗中起着关键的作用。Treg细胞能够有效抑制被某些自体抗原活化的CD4/CD8 T淋巴细胞,当机体缺乏Treg时将会导致结肠炎,关节炎,实验性脑脊髓膜炎,I型糖尿病等自身免疫性疾病;在同种异体移植模型中,也有报道Treg可抑制被供体抗原活化的CD4+效应T细胞,延长移植物存活时间。尽管Treg已经广泛应用于自身免疫性疾病、肿瘤以及器官移植的治疗中,然而,在角膜移植领域,尤其是针对角膜移植排斥反应,使用Treg诱导免疫耐受延长植片存活时间的相关研究还处于起步阶段,国内外鲜有文献报道。目的和内容通过TGF? 2-DC体内诱导Treg及Treg过继转移实验,对不同来源Treg细胞的表型、细胞因子表达、体内作用机制以及抗原特异性进行系统研究,以期揭示Treg在角膜移植排斥反应中的重要作用,进一步深入了解角膜移植排斥反应中Treg的作用机制,为临床有效调控、治疗角膜移植排斥反应及其他自身免疫性疾病提供重要的理论依据。方法1.Balb/c小鼠骨髓来源DC在含TGF-?2的培养液中培养,与供体来源(C57BL/6小鼠)的可溶性抗原共孵育后分离纯化。实验动物分别给予PBS,2×106 TGFβ2-DC尾静脉注射,7天后采用免疫磁珠方法分离纯化脾脏来源的表型为CD4+CD25+的Treg细胞。FACS分析不同来源Treg细胞表型和细胞因子水平;RT-PCR检测Foxp3的表达。MLR检测不同来源Treg在体外对活化CD4+CD25-T细胞增殖反应抑制情况,ELISA检测Treg对活化T细胞细胞因子水平的调节作用。2.分离纯化正常小鼠来源Treg(nTreg)或TGFβ2-DC诱导的Treg(iTreg),Balb/c小鼠分别给予PBS,106 Treg静脉注射,3天后进行小鼠同种异体角膜移植术(C57BL/6小鼠)。移植后不同时间点手术显微镜下观察植片存活时间及排斥曲线,移植后3天收集受体脾脏,FACS分析活化CD4+ T淋巴细胞数量、表型变化及T细胞内细胞因子的表达;MLR检测淋巴细胞增殖情况;ELISA检测淋巴细胞分泌IL-2, IL-10, IFN-γ及TGF-β水平;采用特定中和性抗体(anti-IL-10、anti-TGF-β)拮抗抑制性细胞因子,观察Treg抑制作用的具体机制。3.天然Treg或TGFβ2-DC诱导Treg免疫后的Balb/c小鼠接受C57BL/6小鼠来源的或无关第三方(C3H)小鼠来源的同种异体角膜移植术,移植后3天取受体脾脏T细胞为效应细胞,分别以C57BL/6、C3H小鼠脾细胞为刺激细胞,同时加入Treg共孵育。[3H]-TdR掺入法检测细胞增殖情况。结1.TGF-β2 DC注射后能提高小鼠脾脏中CD4+CD25+T细胞的表达数量(P<0.05);其中CD25、CTLA-4、+Foxp3 T细胞比例在iTreg组显著提高,而CD28细胞比例显著下降(P<0.05)。细胞因子方面iTreg组TGF-β表达较其他两组明显升高,提示TGF-β2 DC诱导的iTreg可以促进TGF-β的分泌。nTreg和iTreg组IL-10表达较CD25-T细胞组显著升高(P<0.05)。混合淋巴细胞培养(MLR)结果显示,iTreg对效应T细胞增殖反应的抑制较其他组差异有统计学意义(P<0.05)。2.天然Treg免疫抑制作用以细胞接触机制为主,获得性Treg兼顾了细胞接触和抑制性细胞因子分泌两种模式;获得性Treg注射组分泌TGF-β的浓度水平明显高于其他三组(P<0.01),而TGF-β2 DC组TGF-β水平无明显改变,说明增多的TGF-β来自获得性Treg细胞,而不是DC产生的。3.CD4+CD25+T细胞可以成功获得供体抗原特异性,获取了供体抗原特异性的获得性Treg可以有效延缓植片排斥反应的发生,MLR结果显示获得性Treg对供体抗原的抑制率(IR)为86.7%。结论TGF-β2 DC在体内可以有效扩增CD4+CD25+Tregs的数量及功能,不同来源Treg在细胞表型、细胞因子分泌等方面均有所不同。经TGF-β2 DC诱导产生、以间接方式获取异体抗原特异性的Treg比天然Treg具有更强的抑制功能,可特异性抑制受体对供体抗原的免疫反应,可以更有效地抑制角膜移植排斥反应。具有广阔的临床应用前景。更多还原

【Abstract】 Purpose:To describe the difference of innate Treg and adaptive Treg in cell phenotype, cytokines, antigen specificity and function mechanism and investigate the different effect of innate Treg and adaptive Treg in immunomodulation of murine corneal allograft rejection.Methods:1.iTregs were generated and expanded in vivo by antigen-loaded immature DCs which had been induced by transforming growth factor beta-2 (TGFβ-2) for 5 days. CD4+CD25+Tregs were isolated by 2-step magnetic cell sorting (MACS) and the Treg–specific phenotypes were identified by FACS. Three days after the injection, the difference of the number and phenotype of different source of Treg (nTreg & iTreg) in SPL were analyzed by FACS in mice of difference groups.2.Two kinds of mechanism were used to observe the effects of nTreg and iTreg function. PBS, TGF-β2 DCs, nTregs or iTregs were injected into the murine tail veins. After that, allogeneic corneal transplantation was performed. Observe the grafts’survival rate and the time when rejection occurred.3.Purified and adoptive transfer different resource of Treg. 3 days later, performed corneal transplantation which the receptor were C57BL/6 or C3H mouse, respectively. Antigen-Specific suppression of graft rejection were observed in MLR by Ex Vivo Stimulated CD4+CD25+ Treg Cells.Results:1.TGF-β2 DC can enhance the number of CD4+CD25+T cells in SPL of mice(P<0.05); Foxp3 and CTLA-4 was preferentially expressed by iTreg cells and CD28 expression decreased in adaptive Terg cells. MLR showed that the suppression to the proliferation of effector T cells of iTreg was significantly higher than other groups.2.TGF-β2 DC increased the number and function of CD4+CD25+Tregs in vivo.The corneal allograft rejection was suppressed notably in iTreg injection group. In iTreg injection group, all the allografts remained clear until 60 days after transplantation and there was statistical difference between the four groups analyzed by NPar test(P<0.05). After adoptive Transfer of TGFβ2-DC, nTregs or iTregs , there was an upregulation in the percentage of CD4+CD25+, CD4+CD25+Foxp3+, CD4+CD25+CTLA-4+T cells in SPL 3 days after corneal allograft transplantation, and there was statistical difference between the these groups (P<0.01). Adaptive Tregs were more potent in suppressing effector than innate Treg (P<0.01).3.CD4+CD25+Treg can acquire alloantigen-specific. iTreg acquired donor antigenic specificity with high inhibition ratio to donator antigen at 86.7%.iTreg can prolong grafts’survival time. Compared with the other 4 groups,the survival time of transplanted corneal graft in adaptive Treg groupwas prolonged significantly (P<0.05).Conclusions:In summary, we indicate that iTreg have more suppressive function than nTreg. Alloantibody was an important effector mechanism for corneal allograft rejection. The suppressor regulatory T cells were involved in the induction of ACAID contribution to suppressing corneal allograft rejection which was through their well-established role in delayed-type hypersensitivity (DTH). Antigen-loaded TGF-β2 DCs could increase the number and function of Foxp3+CD4+CD25+Tregs. CD4+CD25+Tregs with indirect allospecificity were more potent than antigen-loaded TGF-β2 DCs or innate CD4+CD25+Tregs in suppressing effector in allograft rejection specific for the same antigen. Our results add a new important perspective for Treg-based immunotherapeutic strategies.更多还原