松茸多糖（TMP-A）体外免疫调节活性研究

发布时间：2018-05-10 08:26

本文选题：松茸多糖 + RAW264.7　；参考：《西华师范大学》2016年硕士论文

【摘要】：真菌多糖作为一类天然高分子化合物,具有免疫调节、抗肿瘤、降血脂、抗辐射、抗溃疡、抗衰老、抗氧化、抗病毒、降血糖、抗突变等复杂多样的生物活性。因其具有上述多种生物活性现已在医药、保健等行业受到广泛关注,国内外不少科研工作者对许多种类的真菌多糖也做了大量相关研究工作,为天然真菌的开发和利用提供了大量理论资料。松茸作为天然真菌具有极高的营养价值和药用价值,其所含成分具有抗肿瘤、免疫调节等生物活性,而其所含多糖成分尤为突出。虽然对松茸及其多糖的研究有不少文献报道,但大多集中在松茸多糖的提取分离纯化,粗提取物的生物活性的研究,而松茸多糖的纯提取物的结构鉴定及其免疫调节,抗肿瘤等生物活性的研究相对较少。本研究于实验前期从松茸子实体里提取分离纯化获得松茸多糖单体(TMP-A),并对其进行了体外免疫调节活性研究。本研究采用CCK-8法检测TMP-A对免疫细胞(Raji细胞、Jurkat细胞、RAW264.7细胞)的增殖效应;碘化丙啶染色流式细胞术检测TMP-A对RAW264.7细胞周期的影响;中性红法检测TMP-A对RAW264.7细胞吞噬能力的影响;经典的Griess Reagent法检测TMP-A对RAW264.7细胞释NO水平的影响;ELISA法检测TMP-A对RAW264.7细胞的细胞因子(TNF-α、IL-6)释放量的影响;实时荧光定量PCR法检测TMP-A对RAW264.7细胞内细胞因子基因(TNF-α、IL-6、IL-1β)及iNOS基因的mRNA相对表达的影响;Western Blot法检测TMP-A对RAW264.7细胞内iNOS及其COX-2蛋白表达的影响。实验结果表明:TMP-A在一定浓度范围内能显著促进免疫细胞(Raji细胞、Jurkat细胞、RAW264.7细胞)的增殖,细胞周期实验结果表明:TMP-A能够促使处于S期、G2/M期的RAW264.7细胞所占百分比的数量增加,G0/G1期所占百分比的数量下降,这与TMP-A能够促使RAW264.7细胞的增殖相吻合。TMP-A在一定浓度范围内能提高RAW264.7细胞的吞噬能力,促进RAW264.7细胞NO及其相关细胞因子(TNF-α、IL-6)的释放。荧光定量PCR检测结果证实TMP-A能提高RAW264.7细胞内细胞因子基因(TNF-α、IL-6、IL-1β)及iNOS基因的mRNA相对表达。Western Blot法检测结果发现:TMP-A对RAW264.7细胞的内参蛋白β-actin的表达的影响基本不变,iNOS及COX-2蛋白的表达量显著增加。综上所述:TMP-A能够通过激活T、B淋巴细胞、巨噬细胞发挥其免疫调节活性。
[Abstract]:As a class of natural polymer compounds, fungal polysaccharides have complex biological activities, such as immune regulation, anti-tumor, anti-lipids, anti-radiation, anti-ulcer, anti-aging, anti-oxidation, anti-virus, hypoglycemic, anti-mutation and other complex biological activities. Because of its various biological activities, it has received extensive attention in medicine, health care and other industries. Many researchers at home and abroad have also done a lot of related research work on many kinds of fungal polysaccharides. It provides a lot of theoretical data for the development and utilization of natural fungi. As a natural fungus, Tricholoma matsutake has high nutritional value and medicinal value, and its components have anti-tumor, immunomodulation and other biological activities, and its polysaccharides are especially prominent. Although there have been many reports on the studies of Tricholoma matsutake and its polysaccharides, most of them focus on the extraction, purification, bioactivity of crude extracts, and the structure identification and immunomodulation of pure polysaccharides from Tricholoma matsutake. There are relatively few studies on anti-tumor biological activities. In this study, the polysaccharides monomers of Tricholoma matsutake were isolated and purified from the fruit body of Tricholoma matsutake in the early stage of the experiment, and their immunomodulatory activity was studied in vitro. In this study, CCK-8 assay was used to detect the proliferation effect of TMP-A on RAW264.7 cells, flow cytometry was used to detect the effect of TMP-A on the cell cycle of RAW264.7, and the effect of TMP-A on the phagocytosis of RAW264.7 cells was detected by neutral red method. The effect of TMP-A on the release of no in RAW264.7 cells was detected by classical Griess Reagent assay and the effect of TMP-A on the release of TNF- 伪 IL-6 in RAW264.7 cells was detected by Elisa. The effect of TMP-A on the relative expression of cytokine gene TNF- 伪 / IL-6 / IL-1 尾 and mRNA of iNOS gene in RAW264.7 cells was detected by real-time fluorescence quantitative PCR assay. The effect of TMP-A on the expression of iNOS and COX-2 protein in RAW264.7 cells was detected by Western Blot assay. The results showed that the proliferation of RAW264.7 cells in Raji cell line was significantly promoted by 1: TMP-A in a certain concentration range. The results of cell cycle test showed that: TMP-A could increase the percentage of RAW264.7 cells in G _ 2 / M phase of S phase, increase the percentage of G _ 0 / G _ 1 phase, and decrease the percentage of G _ 0 / G _ 1 phase. TMP-A can increase the phagocytic ability of RAW264.7 cells and promote the release of no and its related cytokine TNF- 伪 / IL-6 in RAW264.7 cells. The results of fluorescence quantitative PCR showed that TMP-A could increase the relative expression of TNF- 伪 IL-6 and iNOS gene in RAW264.7 cells. Western Blot assay showed that the effect of TMP-A on the expression of 尾 -actin in RAW264.7 cells was almost the same as that of COX-2 eggs. The expression of white increased significantly. It is concluded that: TMP-A can exert its immunomodulatory activity by activating T T B lymphocytes and macrophages.
【学位授予单位】：西华师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R151

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