KPS调控鸡免疫缺陷活性成分的结构解析及分子机制

For poultry immunosuppression disease pathogeny can cause immune cytoreduction, and function decrease, neural-endocrine-immune network disordered. The hypothesis of immune regulation mechanism was suggested that kadsura marmorata polysaccharides(KPS) could promote the NF-kappa B nuclear transcription factors(NF-κB) to secrete by directing toward Toll-like receptor(TLR) on the surface of immune effective cells (such as T lymphocyte,B lymphocyte,etc) and antigen presenting cells (monocytes,macrophages,etc),or the cAMP-PKA path. The target cell of KPS was confirmed by the immune cell reproductive test. This test was detected cell reproductive activity of T lymphocyte, B lymphocyte and peritoneal macrophage from chicken by MTT method.Immunosuppression pathology model was made with the fifteen days-aged chicken. The effect of KPS on the contents of important cell factor as interleukin-1(IL-1), interleukin-2(IL-2), alpha tumor necrosis factor(TNF-α) and gamma interferon(IFN-γ) and cAMP in outside blood from immunosuppression chicken's were detected by ELISA method. The content of NF-κB was detected by colorimetric DNA-binding assay. The structure of regulation immune deficiency active polysaccharide was analyzed by gas chromatography(GC), mass-spectrography(MS), nuclear magnetic resonance (NMR) method and so on. The immune regulation mechanisms and material base of KPS were clarified. And this lay the foundation for prevention and treatment of immunosuppression disease in the poultry.

针对家禽免疫抑制性疾病病原可引起免疫细胞减少、功能降低，导致神经-内分泌-免疫网络紊乱的现状，提出大叶南五味子多糖可以直接作用于免疫效应细胞(细胞毒性T 细胞、B 细胞等)、抗原递呈细胞（单核细胞、巨噬细胞等）上的TLR受体，或经cAMP-PKA路径促进NF-κB核转录因子的分泌而实现免疫调节的假说，用MTT法检测大叶南五味子多糖对雏鸡T淋巴细胞、B淋巴细胞、腹腔巨噬细胞增殖能力的影响，确证其作用的靶细胞；用15日龄雏鸡制备免疫抑制病理模型，应用ELISA方法检测多糖对免疫抑制雏鸡外周血中重要细胞因子如IL-1、IL-2、TNF、IFN等的含量和cAMP含量的影响；用比色分析法检测NF-κB等核转录因子的含量；用GC、MS和NMR等方法对调节免疫缺陷活性成分进行结构解析。以此阐明大叶南五味子多糖的免疫调节作用机制及其物质基础，为其应用于家禽免疫抑制性疾病的预防和治疗奠定基础。

基于禽免疫抑制性疾病导致免疫器官、组织和细胞的破坏和大叶南五味子多糖（Kadsura marmorata polysaccharides，KPS）能提高雏鸡免疫力、ND疫苗抗体效价背景。主要研究了KPS提取、分离、纯化、结构解析及其免疫活性，海蓬子外种皮、白头翁、党参中多糖的提取及抗菌活性，蚌毒灵散复方中药质量控制、肥猪散的抗菌活性。结果：KPS最佳提取条件为提取时间2.4 h、料液比1:28（g/mL）、提取温度为95 ℃，提取率达到3.50 %，糖含量为76.01 %，乙醇分级沉淀得到KPSⅠ、Ⅱ、Ⅲ、Ⅳ、Ⅴ5个组分，分别经Sephadex柱色谱梯度洗脱得到KPSⅠ-1、KPSⅡ-1、KPSⅢ-1、KPSⅣ-6、KPSⅤ-1五个纯多糖。体外免疫活性组分筛选试验显示，各多糖在50~250 μg/mL时均能单独促进鸡T、B、PMφ细胞增殖，对B淋巴细胞增殖作用高于其他细胞；体内试验显示，KPSIII、KPSIV-6各浓度均能显著促进T细胞分泌IFN-γ、IL-2；各多糖均能促进IL-1的分泌。经PC和GC检测KPSⅢ主要由Xyl与GalA组成，分子量为130406，KPSⅢ能显著地促进T、PMφ细胞增殖，还能显著升高PMφ细胞分泌TNF-α、NO的含量。62.5 µg/mL地塞米松可诱导RAW264.7细胞免疫抑制，100µg/mL的KPSIII 能显著促进免疫抑制模型细胞增殖及TNF-α的分泌；这些实验数据为阐明KPS调节免疫活性及其物质基础具有重要意义。此外，用HPLC-DAD方法建立了蚌毒灵散HPLC指纹图谱已获得发明专利授权证书；肥猪散醇提物对链球菌、金葡菌和大肠杆菌的MIC为31.25~62.5mg/mL；白头翁多糖的最佳提取工艺条件为：料液比1:17(g/mL)，提取温度100℃，提取时间6h，提取6次，提取率为5.07%，对金葡菌、大肠杆菌的MIC 均为80.04 mg/mL；海蓬子外种皮多糖的最佳提取工艺条件为提取温度87 ℃、提取时间5 h、料液比1:57(g/mL)，提取量为(13.03±0.63)mg/g，对金葡菌、大肠杆菌的MIC值分别为50、100 mg/mL；党参多糖的最佳提取工艺条件为提取温度80 ℃，提取次数5 次，提取时间5 h，料液比1:40(g/mL)，提取率为22.31 %，对沙门氏菌有一定抗菌活性。为它们进一步研究奠定基础。

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