【作者】 栗志民；

【导师】 张士璀；

【作者基本信息】 中国海洋大学 ， 细胞生物学， 2008， 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。比较免疫学研究表明脊椎动物和无脊椎动物补体系统有着共同的祖先。在过去10年里,在无脊椎动物后口类、甚至原口类中,结构和功能相似的一些重要补体组分如C3和B因子已经得到了鉴定,但有关这些动物补体功能的研究相当有限。文昌鱼(Amphioxus)是介于无脊椎动物和脊椎动物之间的过渡类型,是研究脊椎动物进化的珍贵材料。本文选择了在系统演化上占据重要地位的文昌鱼,对其体液补体介导弧菌溶菌活性进行研究,并对弧菌逃避补体攻击机制进行探讨。本文首次将测定脊椎动物血清补体溶菌活性的标准方法运用到文昌鱼体液免疫研究中,对文昌鱼体液补体的溶菌活性进行研究。通过文昌鱼体液与弧菌相作用,文昌鱼体液能够在体外抑制溶藻胶弧菌HW284(Vibrio alginolyticus HW284)、副溶血弧菌HW458(Vibrio parahaemolyticus HW458)、哈维氏弧菌SF-1(Vibrio harvey SF-1)的生长,存活率分别为42%, 28%和59%。这样的数据在无脊椎动物的后口类尚属首次。扫描电镜观察表明正常文昌鱼体液存在溶菌活性,例如,能溶解溶藻胶弧菌HW284,而经灭活的体液及无菌过滤海水处理的溶藻胶弧菌HW284表面光滑,细胞完整。用热灭活的兔抗人C3血清处理文昌鱼体液后,再与溶藻胶弧菌HW284相作用,体液溶菌活性明显降低,而经免疫前的兔血清或PBS处理的体液,溶菌活性不受影响,此外,体液经热处理(45℃, 30 min)和反复冻融处理后,文昌鱼体液抑制溶藻胶弧菌HW284生长的活性明显降低,其存活率分别为89%和81%,而正常体液(对照组)作用后,弧菌的生长不受影响。这些结果表明补体作用可能是抑制弧菌生长的主要因素。40 mM EGTA (包含4 mM MgCl2)处理的文昌鱼体液没有影响溶菌活性,与正常体液(对照组)相比,具有相似的溶菌活性,其存活率分别为42%和44% ,在EGTA处理组中添加10 mM CaCl2,没有增强体液的溶菌活性,相反,用40 mM EDTA处理文昌鱼体液,体液的溶菌活性消失,其存活率为86%,在EDTA处理中添加4 mM MgCl2,恢复了体液的溶菌活性,这些实验结果表明文昌鱼体液补体介导的溶菌活性依赖Mg2+,而不依赖Ca2+。另外,用补体替代途径的选择性激活剂—酵母聚糖A处理后的文昌鱼体液,再与溶藻胶弧菌HW284相作用,体液溶菌活性明显降低,与未处理的体液相比,在2倍稀释的体液中,其存活率分别为82%和43%,这些实验结果强烈暗示着补体替代途径的激活与文昌鱼体液的溶菌活性相关。值得注意的是,7种弧菌中,其中4种弧菌—河弧菌HW293(Vibrio furnissi HW293)、辛辛那提弧菌HW287(Vibrio cincinnatiensis HW287)、鳗弧菌W-1(Vibrio anguillarum W-1)和哈维氏弧菌Z3G2(Vibrio harveyi Z3G2)能够抵制文昌鱼体液补体介导的溶菌作用。本文首次将脊椎动物血清补体消耗标准测定方法运用到文昌鱼溶菌功能研究中,使用蛋白酶K消化法分别提取辛辛那提弧菌HW287和溶藻胶弧菌HW284的LPS,经12% SDS-PAGE电泳、银染色后,发现辛辛那提弧菌HW287 LPS既有高分子量的O-抗原又有低分子量的O-抗原的梯状带结构,而溶藻胶弧菌HW284几乎没有高分子量的O-抗原带。高分子量的O-抗原缺乏是与溶藻胶弧菌HW284对文昌鱼体液敏感相一致的。而辛辛那提弧菌HW287具有许多高分子量的O-抗原呈现出补体抵制,表明O-抗原的大小与体液抵制之间是正相关的。此外,用辛辛那提弧菌HW287或溶藻胶弧菌HW284处理后的文昌鱼体液,然后与兔红细胞相作用,结果表明2种弧菌对补体都有消耗,但是与辛辛那提弧菌HW287相比,溶藻胶弧菌HW284对补体消耗显著高的水平。所有这些结果都表明高分子量的O-抗原似乎具有双重的作用:它既能够避免激活补体,又能够阻止结合到细菌表面上的补体损伤细菌细胞膜。由此保护了包括辛辛那提弧菌HW287在内的对文昌鱼体液的杀伤产生抵制作用的弧菌。更多还原

【Abstract】 The Phylogeny of the complement system during evolution has become one of the key problems in immunology. Comparative studies have suggested common ancestries of complement system in both vertebrates and invertebrates. In the past decade, the structurally and functionally similar complement components including C3 and factor B, have been identified in the invertebrate deuterostomes and even in the invertebrate protostome. However, the functions of complement in these animals are rather ill-defined. Amphioxus, a cephalochordate, is the closest living relative to vertebrate, and has been widely known as the most important animal to analyze the origin and evolution of vertebrates. Here, Complement-mediated killing of Vibrio species by the humoral fluids of amphioxus is studied that attribute to better understand the phylogeny of the complement system during evolve of chordate. In addition, this study explores Vibrio species escape the mechanisms of complement’s attacktion.In this study, the standard method of measuring bacteriolytic activity of serum of vertebrate is used in the study of humoral fluids bacteriolytic activity from amphioxus, the first such report in the invertebrate deuterostomes. Here, when humoral fluids from amphioxus Branchiostoma belcheri was interactive with Vibrio species, it was showed that the humoral fluids in vitro were capable of inhibitting growth of some Vibrio species including V. alginolyticus HW284, V. parahaemolyticus HW458 and V. harvey SF-1, only 42%, 28% and 59% of V. alginolyticus HW284, V. parahaemolyticus HW458 and V. harvey SF-1 survived incubation in amphioxus humoral fluids, respectively, the first such data in the invertebrate deuterostomes. Scanning electron microscopy examination definitely revealed the presence of the bacteriolytic activity of amphioxus humoral fluids. V. alginolyticus HW284 incubated in the humoral fluids was lysed, and the cell surface was apparently dissolved, whereas that incubated in the heated humoral fluids and sterilized seawater remained intact, and the cell surface was smooth. Pre-incubation of the heat-inactivated rabbit anti-human C3 serum with amphioxus humoral fluids abrogated the bacteriolytic activity of V. alginolyticus HW284, while pre-incubation with the heat-inactivated non-immune rabbit serum or with PBS did not impair the bacteriolytic activity. This strongly suggested a role of complement in the bacteriolytic activity of amphioxus humoral fluids. This was further supported by the experiments that heat treatment (45℃, 30 min) and repeated thaw and freezing of the humoral fluids markedly reduced the bacteriolytic activity of V. alginolyticus HW284, with 89% and 81% survival observed, respectively, contrasting to 48% survival in the control. 40 mM EGTA plus 4 mM MgCl2 had little effect on the bacteriolytic activity, and survival of V. alginolyticus HW284 (42%) remained comparable to that of the control. In agreement, addition of 10 mM CaCl2 did not enhance the bacteriolytic activity of the EGTA-treated humoral fluids. On the contrary, addition of 40 mM EDTA to the humoral fluids resulted in a significant loss of the bacteriolytic activity, with 86% survival observed, contrasting to 44% survival in the control. On the other hand, the bacteriolytic activity of the EDTA-treated humoral fluids was restored by adding 4 mM MgCl2,This shows strongly the bacteriolytic activity was Mg2+-dependant and Ca2+-independent. In addition, selective activation of the alternative pathway by zymosan A induced a loss of bacteriolytic activity, and resulted in an increased V. alginolyticus HW284 survival. On average, 82% V. alginolyticus HW284 cells survived exposure to a 1: 2 dilution of the humoral fluids previously incubated with zymosan A. In contrast, just 43% of the cells survived in the untreated humoral fluids. Therefore, these results strongly suggest that activation of the alternative complement pathway is responsible for the fluid bacteriolytic activity.It is of note that the four Vibrio species, V. furnissi HW293, V. cincinnatiensis HW287, V. anguillarum W-1, and V. harveyi Z3G2 out of the seven species tested appear resistant to the complement-mediated lysis. Here, the method of consumption of complement monitored in serum of vertebrate is used in the functional study of humoral fluids from amphioxus, the first such data in the invertebrate deuterostomes. LPS was extracted from V. alginolyticus HW284 and V. cincinnatiensis HW287 by a proteinase K method,then subjected to electrophoresis on 12% SDS-PAGE and silver stained. The LPS profiling revealed that V. cincinnatiensis HW287 had an LPS profile with a ladder of both high-molecular-weight (HMW) and low-molecular-weight (LMW) O-antigen bands. In contrast, V. alginolyticus HW284 had few HMW O-antigen bands. Thus the lack of HMW O-antigen bands of V. alginolyticus HW284 coincided with sensitivity to the humoral fluids, whereas the humoral fluid-resistant V. cincinnatiensis HW287 had many HMW O-antigen bands, indicating the presence of a positive correlation between O-antigen size and humoral fluids resistance. Moreover, consumption of complement was monitored by measuring the hemolysis of RaRBC by amphioxus humoral fluids after the fluids had been pre-incubated with V. alginolyticus HW284 or V. cincinnatiensis HW287. It is showed that both Vibrio species tested consumed complement, but V. alginolyticus HW284 consumed significantly higher complement than V. cincinnatiensis HW287, agreeing well with the observation that the humoral fluids were much more bacteriolytic to V. alginolyticus HW284 than to V. cincinnatiensis HW287. All these results suggest that HMW O-antigens may protect the fluid-resistant Vibrio species including V. cincinnatiensis HW287 by a dual act of both avoiding initiating complement activation as well as sterically hindering complement from gaining access to and damaging the cell membrane.更多还原