安络小皮伞多糖的提取及其免疫调节作用研究
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摘要
中医对菌类本草的认识,是依据中医药的理论体系来阐述的,由于菌类本草具有独特的调理先天之本“肾”和后天之本“脾”的作用,所以受到了不少中医药学者的重视与运用。菌类本草中不少药物具有“扶正补虚”的作用,可广泛运用于精神不振、身倦体乏、咳嗽咳血、气短自汗、畏寒肢冷、久病虚羸、舌淡、脉沉细无力等气虚、阳虚、血虚、阴虚、阴阳俱虚的患者。安络小皮伞是一种分布于我国吉林、辽宁等地的担子菌纲真菌,是一种长期被民间食用和药用的菌类本草。本研究以安络小皮伞多糖( Marasmius androsaceus polysaccharide,MAP)粗品为原料,进行免疫活性多糖的提取、纯化并对分离纯化得到的各个安络小皮伞多糖组分进行了免疫活性评价,筛选出具有明显免疫活性的安络小皮伞多糖组分MAP60,在此基础上,就MAP60对免疫抑制小鼠非特异性免疫、特异性免疫功能的影响进行了研究,并采用分子生物学技术手段研究其免疫调节作用机制。
1.安络小皮伞多糖的提取工艺研究
目的:安络小皮伞多糖最佳提取条件的确定。
方法:相关研究表明,影响多糖提取率的主要因素有乙醇浓度、提取温度、提取时间等。为确定安络小皮伞水溶性多糖的最佳提取条件,本实验首先采用乙醇浓度为60%～100%,水提温度为50℃～90℃,水提时间为1 h～5 h,进行单因素试验,以确定提取条件范围。根据单因素实验结果,将醇沉浓度、水提温度、水提时间三项作为考察因素,进行响应面分析,确定最佳提取条件。对提取到的粗多糖继续进行纯化,再对纯化后多糖进行乙醇分级,得到3个安络小皮伞多糖组分:MAP40、MAP60、MAP80。
结果:通过单因素试验和响应面分析,确定了安络小皮伞多糖的最佳提取条件:醇沉浓度为90%、水提温度为80℃、水提时间为5 h。
结论:当采取最佳的提取条件提取安络小皮伞多糖时,此时安络小皮伞多糖得率最高,可以达到51.44%。
2.安络小皮伞多糖免疫活性组分的筛选
目的:对安络小皮伞多糖中具有免疫调节作用的组分进行筛选。
方法:对MAP40、MAP60、MAP80组分进行体外免疫活性测定,分别检测其对T淋巴细胞、B淋巴细胞、腹腔巨噬细胞增殖能力的影响。
结果:MAP40对T淋巴细胞、B淋巴细胞、腹腔巨噬细胞增殖作用不明显,MAP60、MAP80均具有协同有丝分裂原促进T淋巴细胞增殖并提高腹腔巨噬细胞增殖的功能。
结论:MAP60、MAP80是MAP的免疫活性组分。考虑到提取成本及多糖含量等因素,选取MAP60组分继续深入研究其免疫活性。
3.MAP60免疫活性研究
目的:检测MAP60的免疫活性。
方法:采取皮下注射环磷酰胺的方法诱导免疫抑制模型。用非特异性免疫及体液免疫、细胞免疫的相关指标,评价MAP60对环磷酰胺诱导的免疫功能低下小鼠免疫功能的影响。
结果:皮下注射环磷酰胺后,小鼠的非特异性免疫、细胞免疫和体液免疫功能均有所抑制。免疫抑制小鼠腹腔注射MAP60后,MAP60可以增强免疫抑制小鼠非特异性免疫功能如提高吞噬细胞的吞噬功能、提高免疫抑制小鼠脾指数、提高免疫抑制小鼠淋巴细胞水平;增强免疫抑制小鼠的细胞免疫功能如提高迟发超敏反应能力和淋巴细胞转化率;增强免疫抑制小鼠的体液免疫功能如提高抗体分泌水平。
结论:MAP60可以提高免疫抑制小鼠的非特异性免疫、细胞免疫、体液免疫功能,能有效拮抗环磷酰胺对小鼠免疫功能的抑制作用。
4.MAP60免疫调节作用机制的探讨
目的:从分子和基因水平分析MAP60的作用机制。
方法:采用酶联免疫吸附实验对免疫抑制小鼠血清及淋巴细胞培养液中IL-1、IL-2、TNF-α、IFN-γ的分泌水平进行检测,通过反转录聚合酶链反应检测MAP60对免疫抑制小鼠脾细胞中IL-2 mRNA、IFN-γmRNA和腹腔巨噬细胞中IL-1 mRNA、TNF-αmRNA表达的影响。
结果:MAP60可以提高IL-1、IL-2、TNF-α、IFN-γ在外周血中和免疫细胞培养上清液中的水平,提高腹腔巨噬细胞中IL-1mRNA、TNF-αmRNA的表达,提高小鼠脾细胞中IL-2mRNA、IFN-γmRNA的表达。
结论:MAP60在转录水平促进细胞因子的分泌和表达可能是其发挥免疫调节作用的部分机制。
The cognition of traditional Chinese medicine about fungi materia medica is based on the theoretical system of traditional Chinese medicine, because fungi materia medica has the role of regulating the function of kidney and spleen, many researchers pay more attention to it. A lot of fungi materia medica has the function of strengthening body resistance and supplementing body deficiency, it can be widely used to lassitude, burnout, cough hemoptysis, short breath and auto sweat, aversion to cold and limb cold, macronosia and deficiency, pale tongue, fine pulse and asthenia, qi deficiency, yang deficiency, blood deficiency, yin deficiency and yin and yang deficiency patients. Marasmius androsaceus (L.ex Fr.) Fr. is a traditional Chinese medicinal and edible fungus, it distributed in Jilin, Liaoning province of China, which is belonging to the basidiomycetes. This research contents: The study of extraction, purification, immunobiological activities and some of immunoregulation mechanism of Marasmius androsaceus polysaccharides (MAP). The immunomodulating activities of MAP were measured, and an active component of MAP60 was screened out .The effect of MAP60 on non-specific immunity and specific immunity of immunosupressived mice was investigated. The immunoregulation mechanism of MAP60 was studied by molecular biology technique.
1. Extraction technology
Objective: To determine the optimum extraction condition of MAP. Method: Correlation studies indicated that, alcohol concentration, extraction temperature and extraction time are the main influencing factors for polysaccharides extraction. In order to know the range of these extracting conditions, single factor tests were done to analyze the influence of each factor in alcohol concentration (60%~100%), extraction temperature (50℃~90℃), extraction time (1 h~ 5 h). RSA (Response Surface Analysis) experiment design was applied in selecting the optimum combination of these extracting conditions. After a series of purification processes, water soluble MAP was classified by alcohol concentration in three component,MAP40, MAP60, MAP80. Results: By single factor tests and RSA experiment, it is ensured that the optimum extraction alcohol concentration is 90%, the extraction temperature is 80℃, the extraction time is 5 hours. Conclusion: The water soluble polysaccharide is extracted from the Marasmius androsaceus, the extracting rate of polysaccharide is the most (51.44%) at the optimum extracting condition.
2. Selecting the immune activity component of MAP
Objective: To select the immune activity component of MAP from MAP40, MAP60, MAP80. Method: The immune activity of MAP was investigated by the effects of MAP on proliferation of T, B, Mφlymphocyte in vitro study.
Results: MAP60, MAP80 can significantly increase the proliferation of T lymphocytes cell and strongly increases proliferation of Mφ. However, all MAP component had less influence on the proliferation of B lymphocytes cells.
Conclusion: MAP60, MAP80 may contain the immune activity component of MAP. Considering the cost of extracting, we selected MAP60 for further research.
3. Immunocompetence of MAP60
Objective: To study the immunocompetence of MAP60.
Method: Immunosuppressive model was induced by subcutaneous cyclophosphamide (Cy). The effects of MAP60 on the Cy-induced immunosuppressive mice were studied in relation to innate immunity, humoral immunity and cellular immunity parameters.
Results: Cy can inhibit the non-specific immunity, cellular immunity and humoral immunity of mice. After intraperitoneal injected MAP60, the immunity of immunosupressived mice increased. While MAP60 can promote non-specific immunity such as the phagocytic function of macrophage, enhance cellular immunity such as delayed type hypersensitivity and lymphocyte transformation and enhance humoral immunity such as antibody response and secreting.
Conclusion: MAP60 can antagonize the inhibition of non-specific, cellular immunity, humoral immunity in immunosuppressive mice induced by cyclophosphamide.
4. Immunomodulation mechanism of MAP60
Objective: To study the immunomodulation mechanism of MAP60.
Method: The ability of MAP60 to stimulate interleukin-1, interleukin-2, TNF-α, INF-γrelease to peripheral blood and in culture supernatant of mice peritoneal macrophages and spleen cells were assessed by enzyme linked immunoadsorbent assay (ELISA). The gene expression of IL-2 mRNA, IFN-γmRNA, IL-1 mRNA, TNF-αmRNA were assayed by reverse transcription polymerase chain reaction (RT-PCR).
Results: MAP60 can promote the secretion of interleukin-1, interleukin-2, TNF-α, IFN-γin the serum of immunosuppressive mice and promoted the secretion of IL-1 and TNF-αfrom immunosuppressive mouse peritoneal macrophages and promote the secretion of IL-2 and IFN-γfrom immunosuppressive mouse spleen cells. The level of IL-2, IFN-γ, IL-1, TNF-αin test mice were increased after peritoneal injected MAP60, MAP60 can also promote the expression of IL-2 mRNA, IFN-γmRNA, IL-1 mRNA, TNF-αmRNA in immune cells.
Conclusion: It was suggested that the immune regulation effect of MAP60 may be associated with the stimulation of some important cytokine secretion and these cytokine mRNA expression at the transcription level.
1.安络小皮伞多糖的提取工艺研究
目的:安络小皮伞多糖最佳提取条件的确定。
方法:相关研究表明,影响多糖提取率的主要因素有乙醇浓度、提取温度、提取时间等。为确定安络小皮伞水溶性多糖的最佳提取条件,本实验首先采用乙醇浓度为60%～100%,水提温度为50℃～90℃,水提时间为1 h～5 h,进行单因素试验,以确定提取条件范围。根据单因素实验结果,将醇沉浓度、水提温度、水提时间三项作为考察因素,进行响应面分析,确定最佳提取条件。对提取到的粗多糖继续进行纯化,再对纯化后多糖进行乙醇分级,得到3个安络小皮伞多糖组分:MAP40、MAP60、MAP80。
结果:通过单因素试验和响应面分析,确定了安络小皮伞多糖的最佳提取条件:醇沉浓度为90%、水提温度为80℃、水提时间为5 h。
结论:当采取最佳的提取条件提取安络小皮伞多糖时,此时安络小皮伞多糖得率最高,可以达到51.44%。
2.安络小皮伞多糖免疫活性组分的筛选
目的:对安络小皮伞多糖中具有免疫调节作用的组分进行筛选。
方法:对MAP40、MAP60、MAP80组分进行体外免疫活性测定,分别检测其对T淋巴细胞、B淋巴细胞、腹腔巨噬细胞增殖能力的影响。
结果:MAP40对T淋巴细胞、B淋巴细胞、腹腔巨噬细胞增殖作用不明显,MAP60、MAP80均具有协同有丝分裂原促进T淋巴细胞增殖并提高腹腔巨噬细胞增殖的功能。
结论:MAP60、MAP80是MAP的免疫活性组分。考虑到提取成本及多糖含量等因素,选取MAP60组分继续深入研究其免疫活性。
3.MAP60免疫活性研究
目的:检测MAP60的免疫活性。
方法:采取皮下注射环磷酰胺的方法诱导免疫抑制模型。用非特异性免疫及体液免疫、细胞免疫的相关指标,评价MAP60对环磷酰胺诱导的免疫功能低下小鼠免疫功能的影响。
结果:皮下注射环磷酰胺后,小鼠的非特异性免疫、细胞免疫和体液免疫功能均有所抑制。免疫抑制小鼠腹腔注射MAP60后,MAP60可以增强免疫抑制小鼠非特异性免疫功能如提高吞噬细胞的吞噬功能、提高免疫抑制小鼠脾指数、提高免疫抑制小鼠淋巴细胞水平;增强免疫抑制小鼠的细胞免疫功能如提高迟发超敏反应能力和淋巴细胞转化率;增强免疫抑制小鼠的体液免疫功能如提高抗体分泌水平。
结论:MAP60可以提高免疫抑制小鼠的非特异性免疫、细胞免疫、体液免疫功能,能有效拮抗环磷酰胺对小鼠免疫功能的抑制作用。
4.MAP60免疫调节作用机制的探讨
目的:从分子和基因水平分析MAP60的作用机制。
方法:采用酶联免疫吸附实验对免疫抑制小鼠血清及淋巴细胞培养液中IL-1、IL-2、TNF-α、IFN-γ的分泌水平进行检测,通过反转录聚合酶链反应检测MAP60对免疫抑制小鼠脾细胞中IL-2 mRNA、IFN-γmRNA和腹腔巨噬细胞中IL-1 mRNA、TNF-αmRNA表达的影响。
结果:MAP60可以提高IL-1、IL-2、TNF-α、IFN-γ在外周血中和免疫细胞培养上清液中的水平,提高腹腔巨噬细胞中IL-1mRNA、TNF-αmRNA的表达,提高小鼠脾细胞中IL-2mRNA、IFN-γmRNA的表达。
结论:MAP60在转录水平促进细胞因子的分泌和表达可能是其发挥免疫调节作用的部分机制。
The cognition of traditional Chinese medicine about fungi materia medica is based on the theoretical system of traditional Chinese medicine, because fungi materia medica has the role of regulating the function of kidney and spleen, many researchers pay more attention to it. A lot of fungi materia medica has the function of strengthening body resistance and supplementing body deficiency, it can be widely used to lassitude, burnout, cough hemoptysis, short breath and auto sweat, aversion to cold and limb cold, macronosia and deficiency, pale tongue, fine pulse and asthenia, qi deficiency, yang deficiency, blood deficiency, yin deficiency and yin and yang deficiency patients. Marasmius androsaceus (L.ex Fr.) Fr. is a traditional Chinese medicinal and edible fungus, it distributed in Jilin, Liaoning province of China, which is belonging to the basidiomycetes. This research contents: The study of extraction, purification, immunobiological activities and some of immunoregulation mechanism of Marasmius androsaceus polysaccharides (MAP). The immunomodulating activities of MAP were measured, and an active component of MAP60 was screened out .The effect of MAP60 on non-specific immunity and specific immunity of immunosupressived mice was investigated. The immunoregulation mechanism of MAP60 was studied by molecular biology technique.
1. Extraction technology
Objective: To determine the optimum extraction condition of MAP. Method: Correlation studies indicated that, alcohol concentration, extraction temperature and extraction time are the main influencing factors for polysaccharides extraction. In order to know the range of these extracting conditions, single factor tests were done to analyze the influence of each factor in alcohol concentration (60%~100%), extraction temperature (50℃~90℃), extraction time (1 h~ 5 h). RSA (Response Surface Analysis) experiment design was applied in selecting the optimum combination of these extracting conditions. After a series of purification processes, water soluble MAP was classified by alcohol concentration in three component,MAP40, MAP60, MAP80. Results: By single factor tests and RSA experiment, it is ensured that the optimum extraction alcohol concentration is 90%, the extraction temperature is 80℃, the extraction time is 5 hours. Conclusion: The water soluble polysaccharide is extracted from the Marasmius androsaceus, the extracting rate of polysaccharide is the most (51.44%) at the optimum extracting condition.
2. Selecting the immune activity component of MAP
Objective: To select the immune activity component of MAP from MAP40, MAP60, MAP80. Method: The immune activity of MAP was investigated by the effects of MAP on proliferation of T, B, Mφlymphocyte in vitro study.
Results: MAP60, MAP80 can significantly increase the proliferation of T lymphocytes cell and strongly increases proliferation of Mφ. However, all MAP component had less influence on the proliferation of B lymphocytes cells.
Conclusion: MAP60, MAP80 may contain the immune activity component of MAP. Considering the cost of extracting, we selected MAP60 for further research.
3. Immunocompetence of MAP60
Objective: To study the immunocompetence of MAP60.
Method: Immunosuppressive model was induced by subcutaneous cyclophosphamide (Cy). The effects of MAP60 on the Cy-induced immunosuppressive mice were studied in relation to innate immunity, humoral immunity and cellular immunity parameters.
Results: Cy can inhibit the non-specific immunity, cellular immunity and humoral immunity of mice. After intraperitoneal injected MAP60, the immunity of immunosupressived mice increased. While MAP60 can promote non-specific immunity such as the phagocytic function of macrophage, enhance cellular immunity such as delayed type hypersensitivity and lymphocyte transformation and enhance humoral immunity such as antibody response and secreting.
Conclusion: MAP60 can antagonize the inhibition of non-specific, cellular immunity, humoral immunity in immunosuppressive mice induced by cyclophosphamide.
4. Immunomodulation mechanism of MAP60
Objective: To study the immunomodulation mechanism of MAP60.
Method: The ability of MAP60 to stimulate interleukin-1, interleukin-2, TNF-α, INF-γrelease to peripheral blood and in culture supernatant of mice peritoneal macrophages and spleen cells were assessed by enzyme linked immunoadsorbent assay (ELISA). The gene expression of IL-2 mRNA, IFN-γmRNA, IL-1 mRNA, TNF-αmRNA were assayed by reverse transcription polymerase chain reaction (RT-PCR).
Results: MAP60 can promote the secretion of interleukin-1, interleukin-2, TNF-α, IFN-γin the serum of immunosuppressive mice and promoted the secretion of IL-1 and TNF-αfrom immunosuppressive mouse peritoneal macrophages and promote the secretion of IL-2 and IFN-γfrom immunosuppressive mouse spleen cells. The level of IL-2, IFN-γ, IL-1, TNF-αin test mice were increased after peritoneal injected MAP60, MAP60 can also promote the expression of IL-2 mRNA, IFN-γmRNA, IL-1 mRNA, TNF-αmRNA in immune cells.
Conclusion: It was suggested that the immune regulation effect of MAP60 may be associated with the stimulation of some important cytokine secretion and these cytokine mRNA expression at the transcription level.
引文
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