BTLA修饰的未成熟树突状细胞对EAE免疫耐受作用及机制的研究

It is difficult to prevent the disease progression of patients with multiple sclerosis (MS). B and T lymphocyte attenuator (BTLA) is the negative costimulator that regulates the activation of T cells, and immature dendritic cells (DC) induce T cell-mediated immune responses and tolerance by varieties of mechanisms. This study bases on our past research about MS and DC. We produce DC with the antigen specificity by pulsed with MOG35-55 antigen. We construct adenovirus-BTLA (pAd-BTLA) with homologous recombination technology, then immature DCs co-culture with pAd-BTLA aiming to get stable DCs modified by pAd-BTLA. After identification of BTLA-DC in vitro,BTLA-DCs transfuse into EAE mouse, then we observe the effect that DCs modified by pAd-BTLA inhibit the antigen specificity T cell of EAE mouse. We also study the changes of Th1、Th2、Th17 and Treg cells in EAE mouse, and explore the effect of interaction between BTLA and HVEM expressed in mouse dendritic cells on T cell activation. We research the function of Treg cells and effect of signal pathway. This study explores the effects and mechanisms of immune responses and tolerance treated with DCs modified by pAd-BTLA. We aim to look for a therapeutic method with neural immune specificity, and provide a new therapeutic method to the patients with MS.

有效阻止病情的进展是治疗多发性硬化(MS)的难题。B、T淋巴细胞衰减子(BTLA)能有效抑制T细胞活化，未成熟树突状细胞（DC）通过多种机制诱导T细胞特异性抗原的耐受。本项目基于我们以前对MS和DC研究，拟通过MOG35-55致敏冲击未成熟DC的方法，产生抗原特异性DC，采用基因同源重组技术，构建重组腺病毒表达载体(pAd-BTLA)，抗原特异性DC与Adv-BTLA共培养后进行修饰，得到稳定的Adv-BTLA修饰DC（BTLA-DC），体外鉴定BTLA-DC功能后，再输入自身免疫性脑脊髓炎（EAE）鼠内，观察BTLA-DC对EAE鼠的抑制效应，检测T细胞亚群变化，分析BTLA-DC上BTLA与HVEM相互作用对T细胞免疫应答的影响，研究Treg细胞功能和Notch信号通路的变化，阐明BTLA-DC对EAE鼠免疫耐受重建的作用和机制，旨在寻找神经免疫特异性治疗手段，为MS提供一种新疗法。

诱导机体产生特异性免疫耐受被认为是极具前景的治疗多发性硬化（MS）的方法。实验性自身免疫性脑脊髓炎（EAE）是理想的MS研究模型，在EAE发病机制中Th1/Th2细胞分化失衡发挥着重要影响。B、T淋巴细胞衰减子(BTLA)能有效抑制T细胞活化，未成熟树突状细胞（DC）通过多种机制诱导T细胞特异性抗原的耐受。本项目通过MOG35-55致敏冲击未成熟DC的方法，构建重组腺病毒表达载体(pAd-BTLA)，抗原特异性DC与Adv-BTLA共培养后进行修饰，得到稳定的Adv-BTLA修饰DC（BTLA-DC），体外鉴定BTLA-DC功能后，再输入EAE鼠内，观察BTLA-DC对EAE鼠的抑制效应，检测T细胞亚群变化。本研究成功构建携带BTLA基因的重组慢病毒载体，目的基因为BTLA、启动子为CMV，标志基因为可表达新型绿色荧光蛋白的eGFP。qPCR检测重组慢病毒的滴度为3.25 X 109 TU／ml。原代分离培养C57BL/6小鼠骨髓源性树突状细胞。携带BTLA基因的重组慢病毒载体转染C57BL/6小鼠骨髓源性树突状细胞后，经RT-PCR检测发现与单纯树突状细胞的对照组相比，转染了重组慢病毒的树突状细胞的BTLA基因转录水平明显提高（P<0.05)，采用流式检测鉴定其仍为典型树突状细胞表型。成功构建了C57BL小鼠EAE模型。成功分选出EAE小鼠发病高峰时脾细胞中的CD4+T细胞并与树突状细胞建立共培养体系。共培养72小时后经ELISA检测发现，与阴性对照组及空白对照组相比，过表达BTLA蛋白的树突状细胞与EAE小鼠CD4+T细胞共培养体系上清中Th1类细胞因子IFN-γ含量明显减低（P<0.05)，Th2类细胞因子IL-10含量明显增高（P<0.05)。本研究阐明BTLA-DC对EAE鼠免疫耐受重建的作用和机制，为MS提供一种新疗法。