本文選題：香菇多糖 + 腸道粘膜免疫�。� 參考：《廣州中醫(yī)藥大學(xué)》2012年碩士論文

【摘要】：香菇多糖(Lentinan, LNT)具有廣泛的生物學(xué)活性,包括免疫調(diào)節(jié)、抗腫瘤、抗炎、抗病毒,等等。其中LNT的免疫調(diào)節(jié)作用是其生物活性的重要基礎(chǔ)。研究表明,LNT是典型的T細(xì)胞激活劑,通過在體內(nèi)誘導(dǎo)巨噬細(xì)胞產(chǎn)生多種生物活性因子,促進(jìn)T細(xì)胞的增殖活化,增強(qiáng)Th和Tc細(xì)胞的功能,導(dǎo)致一系列免疫應(yīng)答的發(fā)生。 目前,LNT已在抗腫瘤、抗炎等臨床治療中被廣泛應(yīng)用,而且也被研制開發(fā)為各種天然保健品。但是由于多糖難以被機(jī)體降解吸收,關(guān)于口服多糖發(fā)揮功效的機(jī)制尚不明確,具體這類大分子物質(zhì)是如何進(jìn)入機(jī)體發(fā)揮作用的,學(xué)界也存在各種不同的看法。 近年來,對腸道粘膜免疫的研究日益受到重視并逐漸深入,腸道粘膜免疫系統(tǒng)(Intestinal Mucosal Immune System, IMIS)能選擇性地對飲食中的抗原物質(zhì)產(chǎn)生耐受,對病原微生物抗原產(chǎn)生免疫應(yīng)答,并能通過淋巴細(xì)胞的歸巢和再循環(huán)影響機(jī)體整體免疫。IMIS也是口服LNT最先接觸的場所,既往研究表明,LNT能顯著改善免疫抑制小鼠腸道粘膜免疫功能障礙,包括增強(qiáng)T細(xì)胞對外界抗原的敏感性,恢復(fù)淋巴細(xì)胞亞群的正常比例,并且對小鼠整體免疫紊亂和抑制狀態(tài)均有相應(yīng)改善。 綜上所述,本課題將以香菇多糖(LNT)為代表,研究其對腸道粘膜免疫系統(tǒng)T細(xì)胞亞群的影響,為研究中藥多糖發(fā)揮免疫藥理作用的靶點(diǎn)奠定基礎(chǔ)。 研究目的： 建立并優(yōu)化淋巴細(xì)胞內(nèi)細(xì)胞因子流式細(xì)胞術(shù)檢測的實驗條件,在遲發(fā)型超敏反應(yīng)(DTH)小鼠模型基礎(chǔ)上,比較LNT對不同部位各T細(xì)胞亞群的影響；以及采用環(huán)磷酰胺誘導(dǎo)的免疫抑制小鼠模型,初步探討LNT對不同部位αβT細(xì)胞和γδ T細(xì)胞的影響,從上述兩方面探討LNT對粘膜免疫的潛在作用。 研究方法： 1.細(xì)胞內(nèi)細(xì)胞因子檢測的條件優(yōu)化 利用流式細(xì)胞術(shù)建立小鼠淋巴細(xì)胞內(nèi)細(xì)胞因子檢測的方法,以CD3/CD69作為評價指標(biāo),對實驗條件進(jìn)行優(yōu)化,包括刺激劑濃度的選擇和蛋白質(zhì)轉(zhuǎn)運(yùn)阻斷劑的比較選擇。通過對比不同濃度(25、50、75ng/ml)佛波酯(PMA)與離子霉素(Ionomycin)共刺激淋巴細(xì)胞5h后的細(xì)胞因子分泌量,確定PMA的最優(yōu)濃度;通過比較兩種蛋白質(zhì)轉(zhuǎn)運(yùn)阻斷劑(BFA/Monensin Mixture和Monensin)對T細(xì)胞膜表面和胞內(nèi)CD69陽性表達(dá)率的影響,選擇適合的蛋白質(zhì)轉(zhuǎn)運(yùn)阻斷劑。 2.DTH小鼠模型的復(fù)制及LNT對DTH小鼠不同部位T細(xì)胞亞群比例變化的影響 以二硝基氟苯(DNFB)誘導(dǎo)復(fù)制遲發(fā)型超敏反應(yīng)(DTH)小鼠模型,觀察小腸派氏結(jié)(PPs)、腸系膜淋巴結(jié)(MLN)、脾臟(SP)和耳后淋巴結(jié)(RLN)中T細(xì)胞表面活化分子CD69表達(dá)的情況、CD4+/CD8+T細(xì)胞、以及Th1/Th2和Tcl/Tc2細(xì)胞亞群的變化；灌服LNT(800mg/kg)10天后,觀察LNT對DTH小鼠耳腫脹度及上述不同部位T細(xì)胞亞群的影響。 3.LNT對免疫抑制模型小鼠不同部位T細(xì)胞的影響 灌服LNT800mg/kg,7天,采用環(huán)磷酰胺免疫功能抑制小鼠模型,利用流式細(xì)胞術(shù),觀察LNT對SP、PPs和MLN中αβ和γδ T細(xì)胞亞群的影響。結(jié)果： 1.75ng/ml PMA與lonomycin共刺激淋巴細(xì)胞5h后的IFN-y和IL-4的表達(dá)量明顯高于其他兩個濃度；阻斷劑BFA/Monensin Mixture (BM)對T細(xì)胞膜表面CD69表達(dá)的阻斷明顯強(qiáng)于Monensin (MN),BM組CD69表達(dá)率與為刺激對照相當(dāng)；且BM組的胞內(nèi)CD69表達(dá)也高于MN組,以上均表明阻斷劑BM能更有效阻斷胞內(nèi)蛋白向膜外表達(dá)。 2.以DNFB誘導(dǎo)的DTH小鼠,各部位T細(xì)胞活化早期標(biāo)志CD69的表達(dá)顯著性升高;SP與RLN中CD4/CD8比值顯著下降,PPs中CD4/CD8比值顯著上升：而MLN各指標(biāo)無顯著性差異：DTH小鼠四個部位Th與Tc細(xì)胞亞群呈不完全同步的變化趨勢,主要以Tcl (CD8+IFN-γ+)顯著升高為特征。 3.灌服香菇多糖10天后,能減輕DTH小鼠的耳腫脹度：SP與PP中CD4/CD8比值顯著下降,MLN中CD4/CD8比值顯著上升,而RLN各指標(biāo)無顯著性差異;四個部位Th與Tc細(xì)胞亞群的變化,主要以SP中IL-4的降低、局部以RLN和MLN中IFN-γ和IL-4升高為主要特征。 4.腹腔注射環(huán)磷酰胺(CY)100mg/kg,24h后觀察到CY能使各部位αβ T細(xì)胞比例升高,灌服香菇多糖后,PPs和MLN中αβ較CY組顯著升高,而PPs中γδ T細(xì)胞比例恢復(fù)至與正常組相當(dāng)。 結(jié)論： 1.通過胞內(nèi)因子檢測的條件優(yōu)化,確定75ng/ml PMA+1μg/ml lonomycin共刺激淋巴細(xì)胞5h,并選擇BFA/Monensin Mixture為有效的蛋白質(zhì)轉(zhuǎn)運(yùn)阻斷劑。 2.不管是DTH模型還是免疫抑制模型,均屬于免疫功能紊亂的實驗?zāi)Ｐ�。在上述模型�?LNT對各部位T細(xì)胞亞群的作用雖然存在差異性,但對PPs和MLN的作用更明顯,而且對免疫功能的紊亂具有調(diào)節(jié)和恢復(fù)的作用,并推測香菇多糖可能與T細(xì)胞在各部位中的重新分配有關(guān),并對粘膜免疫系統(tǒng)一定的選擇性。
[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.
【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王一心,李梅君;香菇多糖的藥理作用[J];大理學(xué)院學(xué)報;2002年04期

2 劉莉,張軍,方超平,楊佳薈,李聞捷,沈茜;流式細(xì)胞術(shù)檢測人和小鼠Th細(xì)胞亞群[J];第三軍醫(yī)大學(xué)學(xué)報;2004年20期

3 賴先陽;γδT細(xì)胞的發(fā)育與其信號傳導(dǎo)[J];國外醫(yī)學(xué)(免疫學(xué)分冊);2003年05期

4 申蓉,李麗珍;CD69與免疫功能的調(diào)節(jié)[J];國外醫(yī)學(xué)(免疫學(xué)分冊);2004年01期

5 葉月芳;厲有名;;腸上皮內(nèi)γδ T細(xì)胞[J];國際消化病雜志;2008年03期

6 潘冉;香菇多糖臨床應(yīng)用研究進(jìn)展及香菇的藥用價值[J];海峽藥學(xué);2005年04期

7 劉占舉;郭惠學(xué);胡軍;李富廣;杜英;;IL-12對炎癥性腸病患者外周血和腸黏膜固有層淋巴細(xì)胞激活和炎癥介質(zhì)分泌的影響[J];鄭州大學(xué)學(xué)報(醫(yī)學(xué)版);2006年05期

8 楊旭，萬克青，鄭宣鶴，陳惠明，錢煥英，，唐曉鵬，龔國忠，朱映華，何艷;香菇多糖對慢性乙肝患者T細(xì)胞亞群和IL－2受體表達(dá)的影響[J];臨床內(nèi)科雜志;1994年04期

9 郝鈺,邱全瑛,吳s

本文編號：2048668