香菇多糖对小鼠肠道粘膜免疫系统T细胞亚群的影响

发布时间：2018-06-21 12:49

本文选题：香菇多糖 + 肠道粘膜免疫　；参考：《广州中医药大学》2012年硕士论文

【摘要】：香菇多糖(Lentinan, LNT)具有广泛的生物学活性,包括免疫调节、抗肿瘤、抗炎、抗病毒,等等。其中LNT的免疫调节作用是其生物活性的重要基础。研究表明,LNT是典型的T细胞激活剂,通过在体内诱导巨噬细胞产生多种生物活性因子,促进T细胞的增殖活化,增强Th和Tc细胞的功能,导致一系列免疫应答的发生。目前,LNT已在抗肿瘤、抗炎等临床治疗中被广泛应用,而且也被研制开发为各种天然保健品。但是由于多糖难以被机体降解吸收,关于口服多糖发挥功效的机制尚不明确,具体这类大分子物质是如何进入机体发挥作用的,学界也存在各种不同的看法。近年来,对肠道粘膜免疫的研究日益受到重视并逐渐深入,肠道粘膜免疫系统(Intestinal Mucosal Immune System, IMIS)能选择性地对饮食中的抗原物质产生耐受,对病原微生物抗原产生免疫应答,并能通过淋巴细胞的归巢和再循环影响机体整体免疫。IMIS也是口服LNT最先接触的场所,既往研究表明,LNT能显著改善免疫抑制小鼠肠道粘膜免疫功能障碍,包括增强T细胞对外界抗原的敏感性,恢复淋巴细胞亚群的正常比例,并且对小鼠整体免疫紊乱和抑制状态均有相应改善。综上所述,本课题将以香菇多糖(LNT)为代表,研究其对肠道粘膜免疫系统T细胞亚群的影响,为研究中药多糖发挥免疫药理作用的靶点奠定基础。研究目的：建立并优化淋巴细胞内细胞因子流式细胞术检测的实验条件,在迟发型超敏反应(DTH)小鼠模型基础上,比较LNT对不同部位各T细胞亚群的影响；以及采用环磷酰胺诱导的免疫抑制小鼠模型,初步探讨LNT对不同部位αβT细胞和γδ T细胞的影响,从上述两方面探讨LNT对粘膜免疫的潜在作用。研究方法： 1.细胞内细胞因子检测的条件优化利用流式细胞术建立小鼠淋巴细胞内细胞因子检测的方法,以CD3/CD69作为评价指标,对实验条件进行优化,包括刺激剂浓度的选择和蛋白质转运阻断剂的比较选择。通过对比不同浓度(25、50、75ng/ml)佛波酯(PMA)与离子霉素(Ionomycin)共刺激淋巴细胞5h后的细胞因子分泌量,确定PMA的最优浓度;通过比较两种蛋白质转运阻断剂(BFA/Monensin Mixture和Monensin)对T细胞膜表面和胞内CD69阳性表达率的影响,选择适合的蛋白质转运阻断剂。 2.DTH小鼠模型的复制及LNT对DTH小鼠不同部位T细胞亚群比例变化的影响以二硝基氟苯(DNFB)诱导复制迟发型超敏反应(DTH)小鼠模型,观察小肠派氏结(PPs)、肠系膜淋巴结(MLN)、脾脏(SP)和耳后淋巴结(RLN)中T细胞表面活化分子CD69表达的情况、CD4+/CD8+T细胞、以及Th1/Th2和Tcl/Tc2细胞亚群的变化；灌服LNT(800mg/kg)10天后,观察LNT对DTH小鼠耳肿胀度及上述不同部位T细胞亚群的影响。 3.LNT对免疫抑制模型小鼠不同部位T细胞的影响灌服LNT800mg/kg,7天,采用环磷酰胺免疫功能抑制小鼠模型,利用流式细胞术,观察LNT对SP、PPs和MLN中αβ和γδ T细胞亚群的影响。结果： 1.75ng/ml PMA与lonomycin共刺激淋巴细胞5h后的IFN-y和IL-4的表达量明显高于其他两个浓度；阻断剂BFA/Monensin Mixture (BM)对T细胞膜表面CD69表达的阻断明显强于Monensin (MN),BM组CD69表达率与为刺激对照相当；且BM组的胞内CD69表达也高于MN组,以上均表明阻断剂BM能更有效阻断胞内蛋白向膜外表达。 2.以DNFB诱导的DTH小鼠,各部位T细胞活化早期标志CD69的表达显著性升高;SP与RLN中CD4/CD8比值显著下降,PPs中CD4/CD8比值显著上升：而MLN各指标无显著性差异：DTH小鼠四个部位Th与Tc细胞亚群呈不完全同步的变化趋势,主要以Tcl (CD8+IFN-γ+)显著升高为特征。 3.灌服香菇多糖10天后,能减轻DTH小鼠的耳肿胀度：SP与PP中CD4/CD8比值显著下降,MLN中CD4/CD8比值显著上升,而RLN各指标无显著性差异;四个部位Th与Tc细胞亚群的变化,主要以SP中IL-4的降低、局部以RLN和MLN中IFN-γ和IL-4升高为主要特征。 4.腹腔注射环磷酰胺(CY)100mg/kg,24h后观察到CY能使各部位αβ T细胞比例升高,灌服香菇多糖后,PPs和MLN中αβ较CY组显著升高,而PPs中γδ T细胞比例恢复至与正常组相当。结论： 1.通过胞内因子检测的条件优化,确定75ng/ml PMA+1μg/ml lonomycin共刺激淋巴细胞5h,并选择BFA/Monensin Mixture为有效的蛋白质转运阻断剂。 2.不管是DTH模型还是免疫抑制模型,均属于免疫功能紊乱的实验模型。在上述模型下,LNT对各部位T细胞亚群的作用虽然存在差异性,但对PPs和MLN的作用更明显,而且对免疫功能的紊乱具有调节和恢复的作用,并推测香菇多糖可能与T细胞在各部位中的重新分配有关,并对粘膜免疫系统一定的选择性。
[Abstract]:Letinous edodes polysaccharide (Lentinan, LNT) has extensive biological activity, including immune regulation, anti-tumor, anti-inflammatory, antiviral, and so on. The immunoregulation effect of LNT is an important basis for its biological activity. The study shows that LNT is a typical T cell activator. It can induce a variety of bioactive factors by inducing macrophages in the body to promote T cells. Proliferation and activation enhance the function of Th and Tc cells, leading to a series of immune responses.
At present, LNT has been widely used in anti-tumor, anti-inflammatory and other clinical treatment, and has also been developed as a variety of natural health care products. But because the polysaccharide is difficult to be degraded and absorbed by the body, the mechanism about the efficacy of oral polysaccharide is not clear. How this kind of macromolecular substance enters the body, and there are various kinds of studies in the academic circle. Different views.
In recent years, the study of intestinal mucosal immunity has been paid more and more attention and gradually deepened. The intestinal mucosal immune system (Intestinal Mucosal Immune System, IMIS) can selectively tolerate the antigen substance in the diet, produce an immune response to the pathogenic microorganism antigen, and can affect the whole body through the homing and recirculation of the lymphocyte. Immune.IMIS is also the first place to take the first exposure to LNT. Previous studies have shown that LNT can significantly improve the intestinal mucosal immune dysfunction in immunosuppressed mice, including enhancing the sensitivity of T cells to the external antigen, restoring the normal proportion of lymphocyte subsets, and improving the overall immune disorder and inhibition of the mice.
To sum up, the study will take letinous edodes polysaccharide (LNT) as the representative to study its effect on the T cell subsets of intestinal mucosal immune system, which will lay the foundation for the study of the target of Chinese Herbal Polysaccharides to play the role of immune pharmacology.
The purpose of the study is:
The experimental conditions of lymphocyte cytokine flow cytometry were established and optimized. On the basis of delayed hypersensitivity (DTH) mouse model, the effects of LNT on T cell subgroups in different parts were compared, and the immunosuppressive mice induced by cyclophosphamide were used to explore the effect of LNT on the different parts of the alpha beta T cells and the gamma delta T cells. The potential role of LNT in mucosal immunity was discussed from the above two aspects.
Research methods:
Optimization of cytokine detection in 1. cells
The method of cell factor detection in mouse lymphocytes was established by flow cytometry. The experimental conditions were optimized with CD3/CD69 as the evaluation index, including the selection of the concentration of stimulants and the comparison of protein transport blockers. By comparing different concentrations (25,50,75ng/ml) of phorbol ester (PMA) and iomycin (Ionomycin) Co irritation The optimal concentration of PMA was determined by the cytokine secretion after 5h cell, and a suitable protein transport blocker was selected by comparing the effects of two protein transport blockers (BFA/Monensin Mixture and Monensin) on the positive expression of CD69 in the membrane surface and intracellular CD69 of T cells.
Replication of 2.DTH mouse model and effect of LNT on the proportion of T cell subsets in different parts of DTH mice
A mouse model of delayed type hypersensitivity (DTH) was induced with two nitrofluoro benzene (DNFB), and the expression of the surface activation molecule CD69 of the T cells in the small intestinal pies junction (PPs), the mesenteric lymph node (MLN), the spleen (SP) and the posterior auricular lymph node (RLN), the changes of CD4+/CD8+T cells, Th1/Th2 and Tcl/Tc2 cell subgroups, were observed for 10 days. The effects of LNT on the ear swelling and the T cell subsets of DTH mice were observed.
Effect of 3.LNT on T cells in different parts of immunosuppressed mice
The effect of LNT on the subsets of alpha and delta T cells in SP, PPs and MLN was observed by taking LNT800mg/kg, 7 days, using cyclophosphamide immunologic function and using flow cytometry.
The expression of IFN-y and IL-4 in 1.75ng/ml PMA and lonomycin was significantly higher than that of the other two concentrations. The blocking agent BFA/Monensin Mixture (BM) blocked the CD69 expression on the surface of the T cell membrane significantly stronger than that of the Monensin. The results showed that blocking agent BM could effectively block the expression of extracellular proteins.
2. in DTH mice induced by DNFB, the expression of CD69 was significantly increased in the early stage of activation of T cells in each part, the CD4/CD8 ratio in SP and RLN decreased significantly, and the CD4/CD8 ratio in PPs increased significantly, but there was no significant difference in the MLN indexes. Marked rise.
3. after taking letinous edodes polysaccharide for 10 days, the ear swelling of DTH mice could be reduced: the CD4/CD8 ratio in SP and PP decreased significantly, and the CD4/CD8 ratio in MLN increased significantly, but there was no significant difference in RLN index. The changes of Th and Tc cell subgroups in four sites were mainly reduced in IL-4 in SP.
4. intraperitoneal injection of cyclophosphamide (CY) 100mg/kg, after 24h, observed that CY could increase the proportion of alpha beta T cells in each part. After perfusion of letinous edodes polysaccharide, the alpha beta in PPs and MLN was significantly higher than that in the CY group, while the proportion of the delta T cells in PPs was restored to the same as that in the normal group.
Conclusion:
1. by optimizing the conditions of intracellular factor detection, 75ng/ml PMA+1 g/ml lonomycin was determined to co stimulate lymphocyte 5h, and BFA/Monensin Mixture was selected as an effective protein transport blocker.
2. whether the DTH model or the immunosuppressive model, all of which belong to the experimental model of immune dysfunction. Under the above model, the effect of LNT on the T cell subsets of various parts is different, but the effect on PPs and MLN is more obvious, and the function of immune function is regulated and restored, and it is presumed that the letinous edodes polysaccharide may be with T cells. The redistribution in various parts is related to the selectivity of mucosal immune system.
【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.5

【参考文献】