【作者】 王冲；

【导师】 孙振钧；

【作者基本信息】 中国农业大学 ， 生态学， 2005， 博士

【摘要】 本研究以动物免疫学、生态学、生物化学为指导思想,以蚯蚓的生态免疫系统为主线,探讨了赤子爱胜蚓的体壁屏障系统(包括皮肤的物理排除作用和随体表粘液分泌排除)和协同防御作用在其免疫系统中的作用。论文主要分为以下几个部分: 第一部分,分析了角质层和表皮在蚯蚓抗菌生态免疫系统中的作用。结果表明:蚯蚓的体壁主要是通过以下途径来抗细菌的侵袭:一是通过角质层的变化(如上胶层突起的增厚、上角层游离面中微绒毛的变长等)直接抵抗微生物的入侵;二是增加表皮中大颗粒粘液细胞和网状细胞的分泌功能,分泌出大量含有粘多糖蛋白液体复合物的粘液,在蚯蚓体表对细菌进行稀释或者起到直接杀菌的作用;三是通过增加小颗粒蛋白细胞的分泌作用,在蚯蚓的表皮中分泌出更多的抵抗疾病的中性粘多糖,从而抵抗进入蚯蚓体表内的细菌。 第二部分,探讨了体表粘液在蚯蚓抗菌生态免疫系统中的作用。结果表明:(1) 蚯蚓的体表粘液具有抑菌作用,当浓度为0.2g/mL时对金黄色葡萄球菌的抑菌圈直径为1.22厘米:其次为大肠杆菌,抑菌圈直径为1.13厘米,对绿脓杆菌的抑菌作用最差,仅为0.98厘米,诱导后蚯蚓体表粘液对金黄色葡萄球菌和大肠杆菌的抑菌作用显著增加,差异极显著(P<0.01),但对绿脓杆菌的抑菌作用没有显著增加。(2) 经过Sephadex G-75,G—50, G-25三步层析从蚯蚓的体表粘液中分离到抗菌肽粗品ESP,再经过反相高效液相色谱纯化后,得到了一种全新的蚯蚓抗菌肽ESP—1。其是由16种氨基酸的50个残基组成,其中丝氨酸(Ser)残基最多,为10个。经基质辅助激光解吸离子化(MALDI)和飞行时间质谱仪(TTOF/MS)相结合的方法测得ESP—1的分子量为5814.32。ESP—1对大肠杆菌、绿脓杆菌和金黄色葡萄球菌的最低抑菌浓度分别为:6.25mg/mL,12.5mg/mL;50mg/mL。(3) 从蚯蚓的体表粘液中分离出具有抗菌活力和溶菌活力的凝集素粗品,含糖量为1.66±0.16 %,并且在细菌诱导后凝集素的含糖量显著提高,为3.75±0.13%,对大肠杆菌和金黄色葡萄球菌的抗菌活力增强。 第三部分,探讨了脂肪及脂肪酸在蚯蚓抗菌生态免疫系统中的作用,结果表明:蚯蚓脂肪的抑菌作用具有选择性,对大肠杆菌的抑菌作用最强,抑菌圈直径为1.32厘米;其次为绿脓杆菌,抑菌圈直径为0.97厘米,但是对金黄色葡萄球菌没有抑菌作用;大肠杆菌诱导后,蚯蚓脂肪对绿脓杆菌和金黄色葡萄球菌的抑菌作用增强,但是对大肠杆菌的抑制作用减弱。由此可见,蚯蚓的脂肪代谢在蚯蚓的生态免疫过程中发挥着重要作用。 第四部分,探讨了多糖在蚯蚓抗菌生态免疫系统中的作用,从蚯蚓的体壁中分离出具有广谱抗菌活性的粘多糖,并通过高效液相色谱——质谱联用仪分析,得出纯化出的两种粘多糖都是由5个分子组成,其中保留时间为5.3分钟的糖的分子量为534,通过质谱图的解谱,可以推断这种糖的组成为:Glu(葡萄糖)-Xyl(木糖)-Glu(葡萄糖)-Xyl(木糖),而保留时间为6分钟的糖的分子量为452,组成可能是Giu(葡萄糖)-Rha(鼠李糖)-Rha(鼠李糖)或Rha(鼠李糖)—Glu(葡萄糖)—Rha(鼠李糖)。保留时间为6.7分钟的分离峰,糖的分子量为678,组成为Glu(葡萄糖)—Glu(葡萄糖)—Glu(葡萄糖)—Xyl(木糖)—Xyl(木糖),另外两种化合物为未知化合物。 第五部分,探讨了体腔液中不同组分的抗菌蛋白在蚯蚓抗菌生态免疫系统中的协同作用。结更多还原

【Abstract】 The effects of antibiotic barrier (physical and mucilaginous elimination) and joint prevention in the antibacterial immunity system of Eisenia fetida were analyzed in this study by applying animal immunology, ecology and biochemistry. The main results of this study were as the followings.The first part of this study researched the influence of bacterium inducement on the ultrastructure integumentary system in Eisenia fetida. Antibacterial function of body wall was conducted by the following way: microorganism was directly resisted through the change of cuticle, such as incrassation of up-cuticle protuberance and increasing length of micro-floccus in the up-cuticle dissociation surface; exudation function of large granular orthochromatic mucous cells and reticulation mucous cells in the epidermis could be enhanced when the Eisenia fetida was induced by E. coli. Moreover, the mucus that contained compound substance of mucopolysaccharide and protein could make the bactericidal function or dilute the microorganism on the surface of earthworm, which could protect earthworm from invasion of microorganism; the content of neutral mucopolysaccharide of antibacterial function could be increased according with the exudation increment of small granular proteinaceous cells in the epidermis, which could resist bacteria invading into the epidermis of earthworm.The second part of this study researched the function of skin secretions in the antibacterial immunity ecosystem of Eisenia Foetida. It was shown: the antibacterial function of earthworm skin secretions to Staphylococcus aureus was the best and the diameter of bacteriostasis circle was 1.22 centimeter. Those to Escherichia coli and Pseudomonas aeruginosa were 1.13 centimeter and 0.98 centimeter respectively. After the earthworm was induced by Escherichia coli, the bacteriostasis influence of earthworm skin secretions to Escherichia coli and Staphylococcus aureus were increased significantly (P<0.01). But the antibacterial function to Pseudomonas aeruginosa was not increased; Earthworm Slime Peptide (ESP) was purified from body surface slime of Eisenia Foetida by gel filtration using Sephadex G-75 and G-50 connected with G-25. And ESP-1 was achieved from ESP by C-18 reversed-phase High Performance Liquid Chromatography (HPLC) techniques. It was also found that the ESP-1 was composed by 50 residues of sixteen different amino acids, among which ten residues was serine. And molecular weight of ESP-1 was 5814.32 D, which was determined by Matrix Assisted Laser Desorption Ionization (MALDl) and Time Of Flight Mass Spectrometry (TOF/MS). The minimum inhibitory concentrations (MIC) of ESP-1 to Pseudomonas aeruginosa and Staphylococcus aureus were 6.25mg/mL, 12.5mg/mL and 50mg/mL respectively. The crude agglutinin was also isolated from earthworm skin secretions. After the earthworm was induced by Escherichia coli, the content of indicans of this agglutinin was increased from 1.66 + 0.16% to 3.75 + 0.13%. And antibacterial activities of the agglutinin to Escherichia coli and Staphylococcus aureus were also increased, but bacteriolytic activities of the agglutinin were decreased.The third part of this study researched fat and its function in the physical antibiotic barrier ofearthworm. It was shown that the antibacterial function of earthworm fat to Escherichia coli was the best and the diameter of bacteriostasis circle was1.32 centimeter. That to Pseudomonas aeruginosa was 0.97 centimeter. There was no bacteriostasis influence of earthworm fat on Staphylococcus aureus. The antibacterial activities of earthworm fat to Pseudomonas aeruginosa and Staphylococcus aureus were increased after earthworms were induced by Escherichia coli. But that to Escherichia coli was decreased after the inducement.The fourth part of this study researched the polysaccharides and its function in the physical antibiotic barrier of earthworm. Mucopolysaccharides that had broad antibacterial activities was isolated from both earthworms and induced earthworms. Both of mucopolysaccharides were composed by five mole更多还原