松茸多糖TMP-A免疫调节活性及其相关分子机制的研究

Polysaccharides are long carbohydrate molecules, of repeating units joined together by glycosidic bonds. They are often linear, but may also be even highly branched. Polysaccharides are often quite heterogeneous, containing slight modifications of the repeating unit. Depending on the structure, these macromolecules can have distinct properties from their monosaccharide building blocks. They may be amorphous or even insoluble in water . Polysaccharides known as "biological response modulators",reflects its significant immunomodulatory activity, such as promoting the proliferation and differentiation of immune cells, activated T cells and B cells, secrete a variety of lymphokines, regulate the balance of nerve - endocrine - immune regulatory network, and promote the DNA, RNA, protein synthesis et al . In recent years, as more and more polysaccharides have been reported to exhibit a variety of biological activities, including anti-tumor, immunostimulation, anti-oxidation etc., the nonstarch polysaccharides have emerged as an important class of bioactive natural products. In many oriental countries, several immunoceuticals composed of polysaccharides have been accepted such as lentinan, schizophyllan and krestin.Cancer, known medically as a malignant neoplasm, is a term for a large group of different diseases, all involving unregulated cell growth . In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the body through the lymphatic system or bloodstream. Cancer is one of the most fatal diseases in human population and one of the most frequent causes of death worldwide.This study aimed to detect the proliferative capacity effect of TMP-A on Normal Liver(Chang Liver) cells and Spleen cells and the phagocytosis effect of Macrophage cells (RAW264.7) in vitro and on S180 sarcoma cell lines under the right armpit of BALB/c mice in vivo; Use the gene chip technology, quantitative PCR, enzyme-linked immunosorbent experimental techniques and protein imprinting technique to study the pharmacological activity and molecular mechanism of TMP-A for the theoretical foundation and application mode.

真菌多糖多具有抗氧化、抗肿瘤、抗病毒和免疫调节等生物学活性，但由于多糖分离纯化困难及其结构复杂，能够得到结晶单体、阐明其结构、具有免疫调节活性且分子机制明确的多糖匮乏。本研究拟以前期研究中获得的松茸多糖结晶单体TMP-A为研究对象，运用细胞生物学技术研究TMP-A对肝细胞和脾细胞的增殖效应以及对巨噬细胞的吞噬效应的影响，结合荷瘤S180小鼠免疫实验，研究TMP-A的免疫活性。通过运用基因芯片、荧光定量PCR技术、酶联免疫实验和免疫印迹技术，研究其对肿瘤坏死因子（TNF-a）、白细胞介素（IL-2、IL-6）、干扰素(IFN)、一氧化氮合酶（iNOS）和集落刺激因子（CSF）等免疫基因转录和翻译水平的效应，以探寻TMP-A免疫活性及其相关分子机制，为真菌多糖的研究开发提供相关科学依据。

一、研究背景.松茸多糖先前的研究多集中于粗多糖或其复合物，而在其混合物或复合物中具体是哪些成分起作用或者是起主要作用，通过何种方式起作用，其作用效果如何，目前尚不清楚。.二、主要研究内容.松茸多糖结晶单体TMP-A的免疫调节活性及其分子机制。.三、重要结果.TMP-A对巨噬细胞RAW264.7、B细胞(Raji细胞株)、 T细胞（Jurkat细胞株）和原代培养脾脏细胞等增殖具有显著地刺激作用。TMP-A能够显著地刺激巨噬细胞RAW264.7内源性NO分泌和吞噬能力，并诱导其由G0/G1期向S期和G2/M期转变。.TMP-A对Hela细胞具有抑制作用，利用激光共聚焦显微镜检测TMP-A诱导细胞凋亡，并利用流式细胞仪检测在10ug/ml时诱导凋亡率为15%、19%和24.1%。.TMP-A对S180实体瘤具有显著的抑制作用，在浓度为80mg/ml其抑瘤率为70.089%。.TMP-A对巨噬细胞RAW264.7表达谱分析，其中1372个基因高表达，并进行了GO分析和KEGG pathway 分析，并结合荧光定量PCR进行了相应结果验证。.利用Cat # QAM-CAA-4000芯片分析了TMP-A对 200个细胞因子的影响，并结合Elisa和Western Blotting实验对相应结果进行验证。.四、关键数据.表达谱分析显示主要涉及MAPK和NF-κB 两个信号通路，其中Ras、MEK1、MEK2、MKP、ERK、PI3K、AKT、PKC、c-Myc、TRAF6和TRAF2等转录因子具有显著上调作用。.蛋白芯片检测显示，具有20倍以上上调的细胞因子有15个，主要包括CD40、CD40L、IL-28、IL-6、IL-1α、MCP-5、iNOS、JAM-A、LOX-1和TNF-α，其中TNF-α上调781.157倍。.五、科学意义：.本项目运用TMP-A结晶单体研究其免疫活性，明确地证实了多糖除了作为生物体的结构成份和提供能量，还具有免疫调节活性。本项目主要研究对巨噬细胞的刺激作用，进一步明确了多糖通过刺激机体固有免疫系统增殖分化、吞噬和分泌内源性NO及细胞因子发挥免疫调节作用，初步阐明了松茸多糖通过NFκB和MAPK途径刺激固有免疫系统诱导细胞因子的分泌，从而抑制肿瘤细胞的增殖及实体瘤的生长，为多糖的免疫调节机制研究提供了理论基础，为多糖的应用开发提供科学依据。