发酵黄芪多糖基于树突状细胞TLR信号通路的肠粘膜免疫增强作用机制研究

Intestinal or gut mucosal immunity is the fist barrier of disease in the body, the professional antigen-processing cells--dendritic cells (DCs) play the important role in this process.In our lab, we have established a practical cultivation methods on DCs in BABL/c mice, and the previous study results suggested that the Fermented Astragalus Polysaccharides (FASP) which were produced by the incubation with the stain FGM9 of lactic acid bacteria isolated from chicken cecum. FASP was positive for DCs maturation. However, whether or not dose this drug have some immunoenhancement on the intestinal mucosal immunity via DCs TLR signal pathways? and whether or not can the drug play some roles for immuno-promotion via DCs TLR signal pathways in the gut epithelial tissue? All of these need further study to explain and verify. In the present study, the objectives are to identify the TLRs of Toll receptors family which can recognize the fermented Astragalus Polysaccharide after the artificial infection of Salmonella in vivo and LPS induction in vitro, the special proteins like CD103c+ on the surface of the DCs related to the intestinal mucosal immunity were detected by Fluorescence in situ hybridization, immunohistochemical staining and flow cytometry. With the evidences to the difference of MAPK-(AP-1)-NF-κB between the MyD88 and the TRIF signal pathway and the results on the changes of DCs antigen processing activity from electron microscopes and qRT-PCR, orw aims are to clarify the mechanism and enhancement of the FASP via TLR signal pathways in dendritic cells corelated gut mucosal immunity.This research can provide some special basis data for development of new immunoenhancement production and for the clarification of the new mechanism of Chinese herbal.

肠粘膜免疫是机体抗病首道防线，起专职抗原递呈的树突状细胞(DCs)在其中发挥着重要作用。课题组已建立了DCs培养技术，发现黄芪经分离于肠道的乳酸菌FGM9发酵后提取的多糖可诱导小鼠骨髓源DCs成熟，其是否能通过调控DCsTLR信号通路增强肠粘膜免疫？黄芪多糖免疫增强是否与肠粘膜DCsTLR信号通路有关？有待深入研究。为此本项目拟通过体内沙门氏菌感染和体外LPS诱导，依据肠粘膜免疫相关组织中DCs表面CD103c+等特征因子，利用原位杂交和流式细胞技术，鉴定易受发酵型黄芪多糖影响的Toll样受体蛋白TLR类型，比较髓样分化因子88和TRIF介导的DCs MAPK信号通路激活转录因子AP-1和NF-κB的差异，结合粘膜免疫相关DCs的抗原提呈功能分析，阐述发酵型黄芪多糖通过调控DCsTLR信号通路对粘膜免疫的增强作用及机理，旨在为中药多糖免疫增强新机制阐述和新型中药免疫增强剂研提供依据。

肠粘膜免疫是机体抗病首道防线，起专职抗原递呈的树突状细胞(DCs)在其中发挥着重要作用。课题组发前期结果黄芪经分离于肠道的乳酸菌FGM9发酵后提取的多糖可诱导小鼠骨髓源DCs成熟的基础上，通过体内免疫抑制和体外LPS 诱导，结合粘膜免疫相关DCs 的抗原提呈功能分析，阐述发酵型黄芪多糖通过调控DCs TLR 信号通路对粘膜免疫的增强作用及机理，旨在为中药多糖免疫增强新机制阐述和新型中药免疫增强剂研发提供依据。.（1）针对发酵黄芪多糖得率低和提取时间长等问题，通过对超声提取的温度、提取时间、料液比和提取功率4个因素响应面分析，发现在保证多糖提取率不减少的前提下，利用超声波提取法能够使得发酵黄芪多糖提取更为省时高效。经DEAE-52和 sephadex-100纯化获得的多糖得率分别为2.89%和2.03%，纯度达93.7%和90.3%，内毒素含量均小于0.01 EU/mL。.（2）细菌发酵会破坏黄芪细胞壁结构，降解纤维素增加多糖，还可通过相关酶作用将小分子转化为多糖，最终致发酵产物中粗多糖含量比生药黄芪有明显升高。理化特性分析结果提示，发酵黄芪多糖的单糖组成发生了一定变化，含结合蛋白而无游离蛋白，分子量分布趋向降低，红外光谱检测多糖特征峰明显，不含三股螺旋结构，抗氧化活性比发酵前更好，多糖的变化与糖代谢通路有关。.（3）发酵的黄芪中提取的多糖比未发酵和菌种中提取的多糖能更大程度地促进小鼠BMDCs成熟，发酵黄芪多糖刺激小鼠BMDC增殖的最佳剂量和时间分别为100μg/mL和24h，抗原呈递和促进T细胞活化的阳性结果明显。发酵黄芪多糖也能促进小鼠骨髓源CD11c DC和CD103+ DC的成熟及其促进T细胞增殖，可能是通过TLR2和TLR4信号通路促进DC成熟，下游相关信号通路为NF-κB、JNK和P38。.（4）发酵黄芪多糖对环磷酰胺诱导的小鼠肠粘膜免疫抑制具有改善作用，可能与调节SIgA的分泌、上调小鼠肠上皮细胞黏附蛋白E-cadherin、紧密连接蛋白Claudin-2和Claudin-7、ZO家族和Cingulin的表达相关，表明黄芪多糖调节肠粘膜免疫损伤与修复肠上皮紧密连接有关。.（5）已发表论文9篇，正在审稿的SCI 3篇；申报发明专利5件，其中获得授权发明专利4件。