丁酸钠对炎症性肠病中不同表型NK细胞功能的影响及机制研究

Recent researches have shown that there are unique subtype natuaral killer(NK) cells in the intestine,named NK-22 cells, and they maybe involved in the pathogenesis of inflammatory bowel disease(IBD).But the pathogenic role of NK cells in IBD and the exact mechanism are still elusive.NKG2D ligand/NKG2D system is key to the function of NK cells. MICA/B is the main NKG2D ligand in human.Our previous study indicated that the gene polymorphysim of MICA/B was associated with Chinese IBD patients, and the expression of MICA/B and NKG2D in the intestinal mucosa were elevated, which suggested that MICA/B/NKG2D system may play a important role in IBD.This recent research is aimed to further investigate the role of different type of NK cells and MICA/B/NKG2D system in the pathogesis of IBD（both in patients and animal model）. Butyrate have been proved to have the ability of upregulating the expression of MICA/B in tumor cells and some inflammatory cells(including T cells) and sensitizing these cells for cytotoxic effets of NK cells. But there are rather few researches focused on the effect of butyrate on the NK cells and the results are inconsistent.Some studies found butyrate can inhibit the cytotoxicity of NK cells and decrease the expression of cell-activating receptors(NCRs) NKp46,NKp30,NKG2D. But other studies showed butyrate had no effects on the cytotoxicity and NKG2D expression of NK cells. Thus, another main objective of this research is to explore the influence of butyrate on the function of NK cells and MICA/B/NKG2D system in IBD patients and colitis model(in vivo and in vitro).

近来发现肠粘膜中存在独特表型（RORγt+NKp46+或NKp44+）NK细胞，命名为NK-22细胞，可能与炎症性肠病（IBD）发病有关。NKG2D配体/NKG2D系统对于NK细胞发挥功能非常重要。MICA/B是人类主要的NKG2D配体。我们前期研究证实MICA/B基因多态性与汉族IBD相关，且UC患者肠黏膜中MICA/B和NKG2D表达增加，本课题拟进一步探讨不同表型NK细胞及MICA/B/NKG2D系统在IBD中的作用（患者及动物模型）。研究表明丁酸钠能上调肿瘤细胞及某些免疫细胞（如T细胞）上MICA/B的表达并能增强NK细胞对靶细胞的杀伤力。但丁酸钠对NK细胞功能及MICA/B/NKG2D系统影响的研究较少且结论不一，因此本课题拟研究丁酸钠对IBD患者及实验性结肠炎小鼠肠黏膜及外周血中不同表型NK细胞功能及MICA/B/NKG2系统的影响（体内及体外实验），旨在探讨丁酸钠的作用机制。

炎症性肠病（inflammatory bowel disease，IBD）是一组慢性非特异性肠道炎症性疾病，免疫异常在IBD的发病中占关键地位。自然杀伤细胞（natural killer cell，NK cell）是天然免疫系统的重要组分，研究表明NK细胞及NKG2D/MICA/B系统与IBD发病相关；丁酸钠作为短链脂肪酸可调控多种免疫细胞，从而发挥抗炎、抗免疫的作用。本研究中，我们对NK细胞及NKG2D/MICA/B系统在IBD中的作用（临床病例、动物模型、细胞实验）、丁酸钠对实验性结肠炎小鼠的作用机制进行了深入研究。. 首先，建立葡聚糖硫酸钠（dextran sulphate sodium salt，DSS）动物模型，双抗体酶联免疫吸附实验（ELISA）检测发现急性结肠炎组织炎性细胞因子明显升高，以Th2类细胞因子（IL-4、IL-6、IL-10）尤为显著；而缓解期各细胞因子均明显下调趋于正常水平；分离结肠粘膜固有层单个核细胞（LPMC），流式细胞术检测发现急性期LPMC中NK细胞所占百分比无明显改变（约为1.8%），NKG2D总表达率显著升高（正常：2%，急性结肠炎：3.2%），NKG2D+NK占NK细胞比例则明显降低（正常：40%，急性结肠炎：10%）；细胞实验发现10pg/ml MICA抗原刺激NK92细胞，Th1、Th17类细胞因子（TNF-α、IFN-γ、IL-17）显著升高；丁酸钠干预急性结肠炎小鼠可提高生存率，降低临床评分，改善结肠炎性改变，ELISA检测发现Th1、Th2、Th17类细胞因子表达均明显下调基本维持正常表达水平。. 结论：NKG2D+NK细胞可以通过分泌TNF-α、IL-17、IFN-γ来调节Th1/Th2/Th17平衡，从而控制炎症；丁酸钠可能通过调节Th1/Th2/Th17类细胞因子，维持平衡从而控制结肠炎。