【作者】 钱国英；

【导师】 许梓荣；

【作者基本信息】 浙江大学 ， 动物营养与饲料学， 2006， 博士

【摘要】 多糖广泛存在于植物、动物和微生物细胞壁和细胞中，是构成生命的三大基本物质之一，也是具有免疫调节活性的生物大分子物质。本项目研究了超微细菌Glaciecola polaris蛋白多糖(PSK)的常规水浸提法中不同料液比、提取温度和时间对多糖得率、粘度和表面张力的影响；同时研究了超声提取对多糖得率、粘度和表面张力的影响。结果表明：提取时间对多糖的提取率存在显著的影响(p＜0.05)，用水溶剂浸提细菌多糖，料水比1：15，提取温度在100℃左右，提取4h—6h，可获得较高的多糖得率，最高的得率可达8.08％，同时提取液粘度和表面张力较低。超声提取能提高PSK的得率，缩短提取时间，降低提取液的粘度和表面张力，有利于降低多糖浓度极化对分离纯化过程的阻力，提高处理量。超声处理条件以选择10-15min、100W功率，提取4h为宜，最高的得率可达8.82％。从体内抑制试验和体外培养巨噬细胞的杀瘤试验对多糖的抑瘤作用进行了研究。体内抑瘤试验采用灌胃方式，计算抑瘤率和生命延长率。体外试验将巨噬细胞与肿瘤细胞共培养，测定NO浓度与细胞毒效应。培养上清液中一氧化氮(NO)浓度采用NO试剂盒检测，细胞毒效应通过MTT比色法检测。用黄嘌呤氧化酶法测定服用鳖组织浆大鼠脑、肝、血中的超氧化物歧化酶(SOD)含量。结果表明，超微细菌Glaciecola polaris的蛋白多糖和细菌菌体对机体肿瘤具有不同程度的抑制作用，且能有效地增强机体对肿瘤的抵抗性，延长生命期。从细胞毒效应来看，超微细菌Glaciecola polaris的蛋白多糖和细菌菌体能明显促使Mφ的活化，从而激发NO合成显著增高，细胞毒效应增强，促进巨噬细胞对病毒感染细胞的杀伤作用，但这种促Mφ的细胞毒作用随蛋白多糖浓度的变化呈现一定的变化，高浓度(100uL)要好于低浓度(50uL)；从细菌菌体中提取的蛋白多糖要好于细菌菌体本身。另外，Glaciecola polaris的蛋白多糖还可显著地提高SOD的活性，而增强机体的免疫能力。从PSK对受低密度脂蛋白(Ox-LDL)和叔丁基氢过氧化物(tbOOH)等氧化物胁迫的巨噬细胞抗氧化能力进行了体外研究。PSK可有效地保护巨噬细胞减轻氧化损伤，且巨噬细胞克隆刺激因子(M-CSF)也有相似的结果。经PSK处理或M-CSF处理的巨噬细胞的成活率极显著地高于未经处理的巨噬细胞(p＜0.01)。经PSK处理的巨噬细胞培养基能显著地提高巨噬细胞克隆刺激因子的分泌(p＜0.05)，并能显著地增加M-CSF的mRNA的表达(p＜0.05)。由此推测，PSK能减轻Ox-LDL和tbOOH对巨噬细胞的氧化损伤的作用，是与促进巨噬细胞对M-CSF的表达，提高M-CSF的分泌量相关联。以水质pH为应激源，探讨超微细菌Glaciecola polaris的蛋白多糖对中华鳖免疫和抗应激能力的影响。结果表明，超微细菌Glaciecola polaris的蛋白多糖对中华鳖幼鳖血细胞吞噬率、血清溶菌活力和杀菌活力有明显促进作用(p＜0.01)，并对应激引起的皮质醇有抑制作用。由此可见，超微细菌Glaciecola polaris的蛋白多糖的添加不仅对酸应激所致的内分泌功能的紊乱有调整作用，而且对酸应激所致的免疫功能低下有保护作用。研究了蛋白多糖对中华鳖生长性能及免疫功能的影响。选用平均体重50-60g的中华鳖幼鳖300只，随机分成10组，试验组在基础饲料中添加0.05％、0.1％的超微细菌Glaciecola polaris的蛋白多糖，或添加0.1％、0.5％的超微细菌菌体粉碎物，对照组以0.5％α-淀粉代替。饲养8周，测定生产性能、免疫器官指数和血清抗氧化性能。结果表明，各试验组的生长比率、免疫器官指数和血清超氧化物歧化酶(SOD)活性，都显著与极显著地高于对照组(P＜0.05-0.01)，并随添加量的增加而增加。饲料系数各试验组显著地低于对照组(P＜0.05)。超微细菌和蛋白多糖的添加对血清溶菌酶活力的影响差异不显著(P＞0.05)。建议超微细菌Glaciecola polaris的蛋白多糖适宜添加量为0.05％。更多还原

【Abstract】 Polysaccharide exists widely in the cell wall of plant, animal, microorganism and epiphyte. As a kind of biologic substance having the action of immune regulation, it can enhance the function of both the nonspecific immunity and the specific immunity.The extracting conditions of polysaccharide from ultramicro-bacteria Glaciecola polaris by water and ultrasonic treatment were studied in this paper. Effects on sample to water, extracting temperature and extracting time for the polysaccharide yield, mucosity and surface tension were observed in normal extraction. The results showed that there were significant differences between polysaccharide yield in different extracting time (p<0.05 ). The optimum extracting conditions were as follow: the rate of sample to water (w: v), the extracting temperature and the extracting time were 1:15, 100℃and 4h~6h respectively. The highest polysaccharide yield could be reach to 8.08%. Ultrasonic treatment was proved to be an efficient method to enhance the rate of polysaccharide yield, decrease the mucosity and the surface tension of solution, which can shorten the extracting time and reduce the thickness resistance to be prone to separating so that more yield can be gain. 100W ultrasonic power and 10-15min ultrasonic treatment would be the optimum conditions extracted by water for 4h, and the t polysaccharide yield probably could be reach to 8.82%.An experiment was conducted to study the polysaccharide extracted from ultramicro-bacteria Glaciecola polaris on tumor immunization, designed by feeding plamasol to observe the inhibiting tumor trial and using MTT to assay macrophage mediated cytotoxicity .The activities of SOD were observed to determine the effect of antiagting. The results showed that polysaccharide had evident inhibitive effect on S180 tumor growth (P<0.05-0.01). Significant increase (P<0.01) was observed at the prolong longetive rate on them. The induction of nitric oxide in murtur macrophages and macrophage-mediated cytotoxicity in polysaccharide group and germ powder were significantly higher than those of control group (P<0.05～0.01), and high concentration (100uL) is better than less. These observations suggest that polysaccharide could be susceptible macrophage to increase NO production and improve the efficiency of killing tumor cells. Significant increase (P<0.01～0.05) was also observed at the SOD activities between treatments and control, which indicated that the polysaccharide could increase humoral immunity.Effect of polysaccharide(PRK), extracted from ultramicro-bacteria Glaciecola polaris was in vitro investigated in protection of oxidatively- injured mouse macrophages stressed by tert-butyl hydroperoxide (tbOOH) or by oxidatively modified low-density lipoprotein (Ox-LDL). The results showed that PSK treatments to protect macrophages from oxidative injury would be effective and macrophage colony-stimulating factor (M-CSF) exhibited some similar effects. The survival rate of PSK-treated mouse peritoneal macrophages or rhM-CSF-pretreated macrophages cells was significantly higher than that of non-PSK-treated or non-M-CSF-pretreated control cells (p<0.01). Peritoneal macrophages-conditioned medium (PM-CM) obtained from PSK-treated mouse peritoneal macrophages exhibited significantly higher colony-stimulating activity than the conditioned medium of non-PSK-treated cells (p < 0.05) and PSK could significantly increase M-CSF mRNA content in mouse peritoneal macrophages (p<0.05). It was speculated that both M-CSF and PSK could protect macrophages from oxidative injury. PSK-treated macrophages could release more M-CSF and M-CSF could alleviate the Ox-LDL-induced or tbOOH -induced injury to mouse peritoneal macrophages. We proposed that effects of PSK were associated with its capability of inducing M-CSF expression.Polysaccharides extracted from ultramicro-bacteria Glaciecola polaris on immune and anti-stress ability in Chinese soft-shelled turtle stressed by pH were investigated. The results showed that the significant increases (P<0.01) were observed at the phagocytic rate of blood cells, serum bacteriolytic activity and bactericidal bacteriolytic activity in polysaccharide groups. We proposed that effects of PSK might regulate the incretion disturbed by acid-stress and enhance the function of immunity.Effect of polysaccharide on promoting the growth capability and enhancing the function of immunity in Chinese soft-shelled turtle (50-60g) is studied by supplying 0.05 %、0.1 % polysaccharide level, or 0.05 %、 0.1 % germ powder in diets. After 8 weeks, a significant increases (P<0.05-0.01) are observed at the growth efficiency, immune tissues index, SOD activity and feed efficiency in polysaccharides groups. No significant effect on serum bacteriolytic activity is observed ( P>0.05) between trial groups and control group. We proposed that the optimum polysaccharide accession should be 0.05 % in diets.更多还原