【作者】 姜元荣；

【导师】 姚惠源；

【作者基本信息】 江南大学 ， 粮食、油脂及植物蛋白工程， 2004， 博士

【摘要】 本论文以脱脂米糠为原料进行免疫活性多糖的提取分离及免疫活性研究。论文对分离纯化得到的各个多糖组分进行免疫活性评价，筛选鉴定出米糠中的活性多糖成分RBP-Ⅱ，在此基础上，对RBP-Ⅱ的分子组成和结构进行鉴定，并采用先进的分子生物学手段研究其免疫调节作用的机理，为米糠的高附加值综合利用拓展出路，填补我国在此方面的空白。论文主要研究内容如下：首次以挤压膨化处理的脱脂米糠为原料，采用水提(高温、低温)以及碱提方式进行米糠多糖HWSRBP、LWSRBP、ASRBP、AISRBP的提取，考察了脱脂米糠中非淀粉多糖的分布情况：研究显示提取温度是影响水溶性米糠多糖得率的显著因素；高温水提取最佳工艺条件下HWSRBP得率为2． 82％，高于国内外相关文献报道；碱提最佳工艺条件下ASRBP得率为2． 32％。对米糠多糖进行初步纯化，去除提取液中的淀粉和蛋白质，并首次采用10％Ca(OH)2沉淀法去除米糠多糖提取液中的植酸，HWSRBP的灰分含量由14％降为1． 3％，米糠多糖纯度得以提高。采用小鼠灌胃实验考察四种形式米糠多糖的免疫调节活性，结果发现HWSRBP免疫调节活性最显著：对小鼠脾脏和胸腺等脏器器官有明显的促进作用：能够增强正常小鼠脾淋巴细胞增殖功能，是正常对照组的三倍以上；能够提高小鼠腹腔巨噬细胞的吞噬指数和吞噬百分率，且具有量效关系。其次是ASRBP组，中、高剂量条件下免疫增强活性显著；LWSRBP组有一定的免疫活性，但与HWSRBP及ASRBP组相比略低；而AISRBP则无显著生物活性。确定以活性最显著、得率最高的HWSRBP为深入研究对象。考察HWSRBP所可能具有的体外免疫活性，发现HWSRBP在体外能够显著增强由Con A诱导的小鼠脾淋巴细胞的增殖，且增殖作用呈现剂量依赖性，确定该方法为分离纯化过程中的活性检测依据；同时发现，各浓度HWSRBP均能增强小鼠腹腔巨噬细胞NO释放，但没有统计学意义；另外，HWSRBP具有显著的抗补体活性，但量效关系不显著，且试剂造成的批间差异影响较大，仅适宜于短周期实验。采用乙醇分级沉淀、Sepharose CL-6B凝胶过滤柱层析、DEAE Sepharose Fast Flow离子交换柱层析对HWSRBP逐步分级纯化，并结合各分离组分不同浓度条件下对小鼠脾淋巴细胞体外增殖的影响比较，分离得到活性最强组分RBP-Ⅱ。经纯度鉴定证明RBP-Ⅱ为均一成分；同时发现PBP及RBP-Ⅱ生物活性不是由于脂多糖(LPS)及酚酸类物质的存在，或者LPS及酚酸类物质的量不足以提供它们如此显著的生物活性。首次运用UV、IR、HPLC、GC、NMR等现代分析手段，对RBP-Ⅱ的理化性质及结构进行研究：HPLC分析PBP-Ⅱ的平均分子量约为191K，GC结果表明RBP-Ⅱ的单糖组成摩尔比为阿拉伯糖：木糖：葡萄糖：半乳糖=1． 37：0． 50：0． 20：1。综合UV、IR、NMR结果推测RBP-Ⅱ可能的重复单位可表示为：江南大学博士学位论文入汀一”)·劲尸CL’以1与一八，。一口3。一八，。一、上·Jz一u一八比一丈立一J)·p一入y卜(1~一3)一日一Gal一(l一3)一日一Gal一(l一3)一a一Ara一(l~3)一a一Ara一(l一3)一。一Ara·(l一3)一日一xyl一(l一N:以和~·-·-·~N少I惩的形式存在。RBP一n为一种未见相关报道的新的米糠多糖。 考察RBP及RBP一H对环磷酞胺(Cy)造成的免疫抑制模型小鼠的免疫调节作用，发现RBP及RBP一H可以使免疫抑制小鼠的免疫脏器器官恢复至接近正常水平;恢复巨噬细胞的吞噬功能，提示米糠多糖对机体非特异性免疫有增强作用;使T淋巴细胞增殖能力和迟发型超敏反应能力恢复并超过正常水平，提示米糠多糖对T淋巴细胞介导的细胞免疫有增强作用;使经过绵羊红细胞抗原刺激后小鼠的抗绵羊红细胞抗体产生能力增强，提示米糠多糖对免疫功能低下小鼠的体液免疫有增强作用。急性毒性实验结果表明，RBP对健康小鼠无急性毒性，属安全低毒。 首次从细胞因子的角度考察RBP及RBP一H对小鼠体内、体外工L一1、IL一2、TNF一。、工FN-Y细胞因子分泌的影响。RBP及RBP一H在体外有显著促进小鼠腹腔巨噬细胞产生TNF-Q、工L一1的作用，有促进小鼠脾淋巴细胞产生IL一2、工FN一Y的作用。口服米糠多糖RBP及RBP一H后可以明显拮抗环磷酞胺导致的免疫抑制，提高IL一1、TNF一a、工FN一Y的分泌，而使工L一2恢复至接近正常水平。 首次采用RT一PCR技术考察RBP及RBP一11对小鼠体内IL一1、IL一2、TNF一a、IFN一Y四种细胞因子的mRNA表达情况，进一步证实了RBP及RBP一H能够在转录水平上促进细胞因子的分泌，确认了RBP的免疫调节功能以及RBP一H是RBP中活性最强组分的结论。米糠多糖通过促进细胞因子的基因表达来促进机体的免疫调节功能可能是其免疫赋活的机制之一。更多还原

【Abstract】 Rice Bran is an abundant, cheap, and safe raw material for the extraction of immunomodulating polysaccharides. The extraction, purification and immunomodulating activities of rice bran polysaccharides were studied in this paper. The immunomodulating activities of each fraction were measured in vivo and in vitro, and an active fraction, RBP-II, was purified. Its molecular composition and structure were characterized. Meanwhile, the immunomodulating mechanism of RBP-II was also investigated.The suitable extraction techniques for water-soluble rice bran polysaccharide (high temperature, HWSRBP; low temperature, LWSRBP) and alkaline-solubb polysaccharide (ASRBP) from extrusion defatted rice bran were established, and the corresponding extraction parameters were also optimized. Rise in extraction temperature was found to greatly increase the crude polysaccharide yield. Under optimum conditions, the yield of HWSRBP was 2.82%, higher than by reported techniques. The yield of ASRBP was 2.32%. The composition of each non-starch polysaccharide fraction was analyzed for the first time. For primary purification, various procedures were used to remove impurities existing therein, such as starch, protein, etc. 10% Ca(OH)2was added to the solution containing the polysaccharide HWSRBP to precipitate phytin ,50 that the ash content of HWSRBP was decreased from 14% to 1.3%.Compared with other type of crude rice bran polysaccharide, HWSRBP has significantly immunomodulating activity .When HWSRBP was orally administered to mice, the spleen and thymus index of mice were greatly increased ; the lymphocyte proliferation degree was increased three times and the phagocyte ratio and phagocyte index were also significantly higher than control group in a dose-dependent manner. ASRBP has immunomodulating activity when orally administered at high and middle dose, while LWSRBP has lower immunomodulating than HWSRBP and ASRBP. AISRBP didn’t had no immunomogolating activity. HWSRBP was studied in depth in the following research for its great biological activities and highest yield.The immunomodulating activity of HWSRBP in vitro was studied. HWSRBP can significantly enhance the proliferation of T lymphocyte induced with Con A in vitro, and is dose dependent. This method was used to detect the biological activity of each purified fraction. HWSRBP can also enhance nitric oxide secretion of peritoneal macrophage of mice, but this effect had no statistical significance. HWSRBP has marked anti-complementary activity, and this method was suitable to detect biological activities of polysaccharides in short periods in view of agent variations.HWSRBP was successively fractionated by stepwise ethanol precipitation, and column chromatography on Sepharose CL-6B and DEAE Sepharose Fast Flow. The effect of each fraction at various concentrations was measured on the proliferation of T lymphocytes induced by Con A in vitro, and an active fraction, RBP-II, was purified. The relationship between the structure and the biological activity of RBP-II was studied. Results indicated that anti-complementary activity of RBP-II was due to polysaccharide rather than LPS and thepulyplionol moieties, feruiic acid, being an possible component in rice bran.The physicochemical properties of RBP-II were studied. Gas Chromatography (GC) shows that RBP-II consist Arabinose, Xylose, Glucose and Galactose in a ratio of 1.37: 0.50: 0.20: 1; Its molecular weight is about 191 000. UV, IR and NMR spectra reveal that RBP-II consist a repeating unit with the following structure:RBP-II is a new polysaccharide isolated and purified from defatted rice bran.RBP and RBP-II can stimulate humoral, cellular and non-specific immunity by oral administration in mice with immunosuppressive agent Cyclophophamide (CY). J.tp. injection of CY can inhibit the proliferation of splenocytes , phagocytic function of PM . While RBP-II can promote non-specific immunity such as the phagocytosis of macrophages, stimulate cellular immunity such as the proliferation of splennocytes and the genesis更多还原