【作者】 王长法；

【导师】 张士璀；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2003， 博士

【摘要】 补体的系统发育进化是当今免疫学研究热点之一。虽然对补体研究已有100多年历史，但补体最早出现于哪类动物及其激活适应系统演化仍不十分清楚。本文选择了在系统演化上占据主要地位的文昌鱼以及软骨鱼和硬骨鱼，对它们的补体系统进行了研究。 本文首次将脊椎动物血清补体溶血活性标准测定方法运用到文昌鱼体液免疫研究中，首次建立了测定无脊椎动物体液补体溶血ACH50的方法。我们发现文昌鱼体液出现补体样的溶血活性。文昌鱼体液可溶解悬浮在10mmol／L EGTA-Mg-GVB缓冲液中的正常的兔血红细胞、绵羊红细胞、及部分哺乳动物、鸟类、两栖类和鱼类的代表动物的血红细胞；而在GVB²⁺缓冲液中时对抗体包被的绵羊红细胞却没有溶血活性。文昌鱼体液的溶血活性和靶红细胞的动物种类在系统发育上和文昌鱼的亲缘关系远近没有直接联系。文昌鱼体液在EGTA-Mg-GVB缓冲液中对兔血红细胞发生溶血的最适条件是：温度是20℃，最适pH是7.5，最适Mg⁺⁺的浓度是10mmol／L。溶血活性是二价离子依赖的，热敏感的（溶血活性热灭活温度是45℃）。文昌鱼体液对兔血红细胞的溶血活性在受到酵母聚糖、甲胺、肼、PMSF、EDTA、兔抗人补体3抗血清处理时，溶血活性消失。此外，本实验还运用免疫比浊法以绵羊抗人的补体3的抗血清检测了文昌鱼体液中的补体3浓度为1.17mg／ml。此浓度和人血清及狗血清中的C3浓度基本相当。这些结果说明：文昌鱼体液中存在着类似于脊椎动物替代途径补体样的溶血活性。 此外，文昌鱼体液中是否存在凝集素及其性质如何一直没有报道。本文运用血细胞凝集试验证实在文昌鱼体液中存在凝集素。同时还首次发现文昌鱼体液中的凝集素活性是SH依赖型，还特异性被多个含D—半乳糖苷的多糖抑制。经注射鸡大肠杆菌后，文昌鱼体液中的凝集素对人B、O型红血细胞，兔、草鱼和蟾蜍红血细胞的凝集活性明显增强。此结果显示，文昌鱼体内可能存在一定获得性免疫反应。 全世界鱼类据估计大约有24，000种左右，其中仅有少数几种鱼类的免疫系统被系统研究过。但在被研究过的少数几种鱼类中，淡水鱼类占了绝大多数，只中国海洋大学博士学位论文中文摘要有一种软骨鱼类一长鳍斑竹鳖的补体系统多年前被研究过。本文运用脊椎动物血清补体溶血活性标准测定方法定性、定量地研究了软骨鱼条纹斑竹鳖的血清溶血补体系统。发现条纹斑竹鳖血清中既存在补体经典溶血途径，又存在补体替代溶血途径。条纹斑竹鳖血清ACH50和CH50值分别为257.2 Units/ml和35.8士13.1Units/ml（n=6）。条纹斑竹鳖血清对悬浮在EGTA一Mg一GVB缓冲液中的正常的兔血红细胞、绵羊红细胞、及部分哺乳动物、鸟类、两栖类和鱼类等代表动物的血红细胞具有溶血活性;在GVBZ十存在时对抗体包被的绵羊红细胞具有溶血活性，而在EDTA一GVB缓冲液中不论是对正常的兔红细胞或其他脊椎动物的红细胞还是对抗体包被的敏感绵羊红细胞都没有溶血活性。此外，条纹斑竹鳖血清对不同脊椎动物红细胞的溶血活性和靶红细胞的动物种类在系统发育上和条纹斑竹鳖的亲缘关系远近没有直接联系。条纹斑竹鳖血清通过补体替代途径对兔血红细胞发生溶血的最适条件是:温度是20℃，最适PH是7.5，最适Ms++的浓度是smM。溶血活性是二价离子依赖的，热敏感的（溶血活性热灭活温度是45℃）。条纹斑竹鳖血清通过补体替代途径对兔血红细胞的溶血活性在受到PMSF、EDTA、酵母聚糖、甲胺、脐处理时，溶血活性消失，将条纹斑竹鳖血清预先经兔抗人C3抗血清处理后，其对兔红细胞的溶血活性部分受到抑制。另一方面，条纹斑竹鳖血清通过补体经典途径发生溶血的特性是:需要同种鱼抗绵羊红细胞抗血清致敏的绵羊红细胞作为靶细胞;需要二价CaZ+和M广离子参与:温度是25℃;作用时间是60分钟;该溶血过程可被特异性抑制剂如L一赖氨酸、角叉藻聚糖、酵母聚糖、甲胺和腆所抑制。实验结果显示:条纹斑竹鳖血清中存在着类似于哺乳动物经典、替代途径补体溶血系统，并且条纹斑竹鳖体内的替代途径补体溶血活性比哺乳动物的要高得多，且其溶血补体系统在低温环境下（如0一4℃）皆可充分激活，显示补体替代溶血途径在条纹斑竹鳖抵御外界病原微生物的侵染时起着重要作用。较高的ACHSO值可以扩大其天然免疫识别能力，弥补体内不成熟的获得性免疫能力。 除软骨鱼之外，我们还运用脊椎动物补体溶血途径的标准检测方法，定性、定量地研究了硬骨鱼草鱼血清溶血补体系统。发现草鱼血清中既存在补体经典溶血途径，又存在补体替代溶血途径，其ACHSO和CHSO值分别为46.9 Units/ml和8.8士5.1 Units/耐（n二6）。草鱼血清可溶解悬浮在缓冲液EGTA一Mg--GVB中的正中国海洋大学博士学位论文中文摘要常的兔血红细胞、绵羊红细胞、及部分哺乳动物、鸟类、两栖类和鱼类的代表动物的血红细胞;而对悬浮在缓冲液EDTA一VB中的这些脊椎动物代表动物的血红细胞却没有显示溶血活性。同时还发现草鱼血清通过激活替代途径的溶血活性大小和靶红细胞的动物种类在系统发育上和草鱼的亲缘关系远近没有直接联系。草鱼血清通过补体替更多还原

【Abstract】 The phylogeny of the complement system during evolution has become one of the key problems in immunology. The studies on complement have been over 100 years, but it remains unclear when and which animal is initially equipped with the ancestral complement system. Here, we report the results of the study on the complement system of the cephalochordate amphioxus and lower vertebrates lip shark and grass carp. This can help us not only betters our understanding of the phylogeny of the complement system during chordate evolution but also provides an important link between innate and adaptive immune responses.Amphioxus or lancelet, a cephalochordate, is the extent invertebrate most closely related to the vertebrates. We have invented a technique to extract the humoral fluid from the amphioxus Branchiostoma belcheri tsingtauense. The presence of complement-like activity in the humoral fluid of amphioxus was examined. The complement classical pathway (CP) activity is assayed by determining the amount of serum required to hemolyse a given number of sensitized sheep red blood cells (SRBC), whereas the complement alternative pathway (AP) activity measured in the same manner, but by using rabbit red blood cells (RaRBC) instead of sensitized SRBC. It was demonstrated that functionally and structurally similar complement components are present in the humoral fluid of amphioxus. The humoral fluid fromamphioxus was capable of causing hemolysis of rabbit erythrocytes and those obtained from species representing mammals, birds, amphibians and fish, but not sensitized sheep erythrocytes. There was no relationship between phylogenetic proximity of erythrocyte species to Branchiostoma and degree of hemolysis. The hemolytic activity was optimally assayed at 20癈, at pH 7.5, and in the presence of 10 mM Mg2+. The hemolytic activity was Mg2+-dependent and heat-sensitive, and was abrogated by treatment with rabbit anti-human C3 serum, zymosan, methylamine, hydrazine, and phenylmethylenesulfonyl fluoride (PMSF). In addition, titration by turbidimetric immunoassay (TIA) using sheep anti-human C3 serum revealed that amphioxus humoral fluid contained C3-like component, and its concentration is about 1.17 mg/ml, which is comparable to C3 concentration in human or dog sera. These suggest that the hemolytic activities displayed by amphioxus humoral fluid appear to represent the vertebrate complement system probably operating via the alternative pathway (AP).The presence of lectins in the humoral fluids of amphioxus Branchiostoma belcheri tsingtauense, was also studied. It was showed for the first time that lectins were present in the humoral fluids of amphioxus. The hemagglutinating activity of the fluids was not affected by treatment with 50 mM EDTA and EGTA, while it was increased by treatment with 3 -ME. Therefore, hemagglutination activity of amphioxus humoral fluids is Ca2+-independent, but SH group-dependent. Hemagglutinating activity of the humoral fluids was inhibited by methyl- β -D-galactopyranoside, thiodigalactoside, D-galactose and asialofetuin. In contrast,N-acetyl-D-galactosamine, sucrose, D-glucose, glycogen, D-mannose, L-fucose and fetuin had no inhibitory effects on hemagglutination. So the lectin activity in the humoral fluids of amphioxus is SH group-dependent and is specifically inhibited by various D-galactosides. Also, the humoral fluids obtained from E. coli-injected amphioxus showed increased agglutinating activity against human B and O, rabbit, grass carp and toad erythrocytes, but not against human A and chick erythrocytes, hinting that there might be two types of lectins in amphioxus humoral fluids.To compare the complement lytic system between cephalochordate and cartilaginous fish, lip shark (Chiloscyllium plagiosum) serum complement activities were investigated. In this part, we reported for the first time the hemolytic activities of lip shark serum via the complement alternative pathway (AP). Lip shark serum was capable of hemolyzing rabbit erythrocytes and those of the vertebrate spe更多还原