【作者】 张朝霞；

【导师】 苏永全；

【作者基本信息】 厦门大学 ， 海洋生物， 2006， 博士

【摘要】 本论文致力于研究病原和非病原细菌的入侵引起杂色鲍(Haliotis diversicolor)血细胞免疫功能和体液免疫因子的变化,从形态与功能对鲍的血细胞进行分类,研究了鲍血细胞吞噬细菌的过程,探讨了鲍血细胞和体液免疫系统对病原弧菌和非病原细菌及其胞外产物(Extracellular products,ECP)的不同防御机制,阐述抗生素对杂色鲍血细胞免疫功能的影响。主要的研究结果包括3个部分:第一部分杂色鲍血细胞的形态观察和功能研究1.首次以血细胞在抗凝剂中的死亡率和凝集情况为指标,对实验室自行配制的3种鲍血淋巴抗凝剂和其它6种无脊椎动物血淋巴抗凝剂进行了筛选。结果表明,在自行配制的抗凝剂HA1和RH中的鲍血细胞分布均匀,6h内死亡率均低于6%,24h死亡率均未超过33%,抗凝效果最佳。2.首次根据血细胞的形态结构结合其细胞核质比和免疫功能特点将杂色鲍的血细胞分成3类:一类血细胞为7.1~8.6μm×7.8~9.2μm,核质比均值1.39,细胞质中一般仅见线粒体以及杆状、椭圆状和中空状的丰富颗粒,可吞噬细菌和酵母,NBT还原反应阴性的颗粒细胞(Ⅰ型);二类血细胞为6.7~8.1μm×7.1~8.3μm,核质比均值1.82,细胞质无颗粒且富含内质网,可吞噬细菌和粘附酵母,具有NBT还原能力的透明细胞(Ⅱ型) ;三类血细胞为4.56~6.49μm×3.82~5.75μm,核质比均值为3.24,细胞质中有较多的线粒体,没有吞噬功能也不还原NBT的类淋巴细胞(Ⅲ型)。第二部分杂色鲍离体血细胞的免疫功能及其影响因子的研究。1.首次研究了2种病原弧菌(F3602和X4302)和非病原大肠杆菌(Escherichia coli)菌液对腹足类离体血细胞产生活性氧、吞噬活性、溶酶体膜的完整性和趋化性4项免疫功能的影响。结果表明,与浓度相同的E.coli菌液相比,2种弧菌抑制了离体杂色鲍血细胞产生活性氧,但差异不显著;严重破坏了血细胞溶酶体膜(P<0.05);菌液浓度同为2.1×107cfu/mL时,血细胞对F3602和X4302的趋化距离分别是E.coli的1.69(P<0.05)和1.57倍;血细胞对浓度为6.3×107cfu/mL的2种病原弧菌的吞噬作用显著大于对同一浓度E.coli的吞噬(P<0.05)。2.首次报道菌ECP对腹足类离体血细胞免疫功能的影响。蛋白浓度为30mg/L的2种弧菌ECP对杂色鲍血细胞溶酶体膜的破坏性远高于E.coli ECP(P<0.05);蛋白浓度同为7.5mg/L,X4302和F3602 ECP导致血细胞的趋化性高于E.coli ECP,其中F3602 ECP组与E.coli ECP组血细胞的趋化性差异显著(P<0.05);蛋白浓度同为30mg/L时,F3602 ECP组血细胞对F3602的吞噬活性明显地大于E.coli ECP组(P<0.05);在同一浓度下,加入2种弧菌ECP后血细胞产生的活性氧少于E.coli ECP组,但差异均不显著。3.首次研究了抗生素对离体贝类血细胞免疫功能的影响。结果表明,与生理盐水对照组相比,链霉素对离体杂色鲍血细胞溶酶体膜破坏作用微弱,略微抑制血细胞的趋化性,提高血细胞活性氧的作用不明显(P>0.3),浓度≧50μg/mL的链霉素显著促进血细胞对F3602的吞噬(P<0.05),可做为养殖杂色鲍弧菌病的防治药物。浓度≧50μg/mL的新霉素显著抑制血细胞趋化(P<0.05),对血细胞吞噬活性的促进作用不如链霉素,不宜用于鲍弧菌病的防治。氯霉素和壮观霉素对血细胞吞噬活性的促进作用不明显,严重破坏溶酶体膜(P<0.05),浓度为100μg/mL时明显抑制血细胞产生活性氧(P<0.05),浓度为100μg/mL和50μg/mL的2种抗生素作用下血细胞的趋化距离均明显缩短(P<0.05),在养殖鲍弧菌病防治中均应被禁用。4.15℃和30℃两组温度条件下,低温组的离体鲍血细胞在F3602及其ECP作用下,相对高温组呈现出溶酶体膜的稳定性加强,产生的活性氧减少,对F3602的吞噬活性增强,血细胞的趋化距离缩小。而在抗生素实验中,相对高温组而言,低温组的离体鲍血细胞产生的活性氧稍有波动,吞噬活性略有下降,除了浓度为100μg/mL的壮观霉素和新霉素组鲍血细胞趋化距离变大以外,其余实验组血细胞的趋化距离缩小,抗生素对溶酶体膜的破坏作用相对减轻。结果表明,低温条件可以减轻抗生素对血细胞溶酶体膜的破坏,更有利于贝类的免疫系统发挥作用。第三部分病原弧菌和E.coli及其ECP感染对杂色鲍活体血细胞的免疫功能和体液免疫因子的影响研究。1.经人工感染实验证实副溶血弧菌F3602及其ECP均可以导致杂色鲍死亡,48h和96h的半致死浓度分别为6.88×10~7cfu/mL、2.84×10~7cfu/mL和252.9mg/L、183.2mg/L。在低于48h半致死浓度的F3602和E.coli及其ECP感染健康杂色鲍后对活体血细胞的免疫功能和体液免疫因子的变化进行研究。对于F3602及其ECP而言,与注射生理盐水的对照组相比,菌液浓度≧8.7×10~7cfu/mL时F3602可导致杂色鲍活体血细胞的溶酶体膜被显著破坏(P<0.05),趋化性被显著抑制(P<0.05),活体血细胞产生的活性氧显著增加(P<0.05),低浓度(5.8×106cfu/mL和8.7×106cfu/mL)F3602菌液显著促进血细胞对F3602的吞噬(P<0.05)。高浓度的F3602 ECP(蛋白浓度为204mg/L)感染明显抑制杂色鲍活体血细胞产生活性氧(P<0.05)和对F3602的吞噬活性(P<0.05),极严重破坏血细胞的溶酶体膜(P<0.01),明显提高血细胞的趋化性(P<0.05)。对于E.coli及其ECP而言,注射高浓度的E.coli菌液明显促进了鲍血细胞对F3602的吞噬(P<0.05),极严重破坏了血细胞的溶酶体膜(P<0.01)。高浓度的E.coli ECP感染仅极显著增加了活体血细胞活性氧的产生(P<0.01)。低浓度的E.coli菌液及其ECP(蛋白浓度为51mg/L)感染对血细胞的上述4项免疫功能几乎没有影响。2.分析了F3602和E.coli感染杂色鲍48小时后血清中碱性磷酸酶(Alkaline phosphatase,ALP)、酸性磷酸酶(Acid phosphatase,ACP)和超氧化物歧化酶(Superoxide dismutase,SOD)的比酶活改变。首次报道了菌ECP感染对贝类血清中的ACP、ALP和SOD酶活力的影响。结果发现,高浓度的F3602 ECP和F3602菌液感染可显著地抑制鲍血淋巴中SOD的活力(P<0.05);鲍血淋巴中ALP的比酶活因注射低浓度的E.coli菌液和高浓度的F3602菌液而明显提高(P<0.05);低浓度的E.coli菌液和低浓度的F3602 ECP(蛋白浓度51mg/L)感染可显著提高鲍血淋巴中ACP的比酶活(P<0.05),ACP比酶活却因注射高浓度的F3602菌液而大大降低(P<0.05)。3.首次报道腹足类血清中存在与羊抗人抗血清发生特异性反应、类似人IgA、IgG和IgM的类抗体。结果表明,细菌感染前杂色鲍血清中类IgA、类IgG和类IgM含量在64～71μg/mL之间,经浓度为8.7×10~6cfu/mL E.coli诱导后鲍血清中类抗体的含量大大增加,IgM为157.008±18.201μg/mL、IgG为100.252±6.870μg/mL、IgA为88.019±6.599μg/mL。细菌感染后IgM含量和其他动物及人在一个数量级,IgA和IgG含量均比其他动物及人低1-2个数量级。4.首次比较了F3602和E.coli菌液及其ECP感染对贝类血清中类抗体含量的影响。低浓度的2种菌液和ECP均可使杂色鲍血清中类IgA的含量显著高于生理盐水对照组(P<0.05);低浓度的2种菌液和浓度为51mg/L的E.coli ECP同样可以显著增加血清中类IgG的含量(P<0.05)。但菌液及ECP对IgM抗体的诱导作用则表现为:低浓度F3602菌液组鲍血清中类IgM含量显著低于同浓度E.coli菌液组(P<0.05),相反,高浓度F3602菌液组鲍血清中的类IgM含量显著地高于同浓度E.coli菌液组(P<0.05)。与对照组相比,高浓度的F3602 ECP组鲍血清的类IgM含量显著下降(P<0.05),低浓度的F3602 ECP和E.coli ECP分别导致血清中类IgM含量极显著增加(P<0.01)和显著增加(P<0.05)。更多还原

【Abstract】 Hemocyte’s function and immune factors in sera of Haliotis diversicolor changed by invasion of pathogenic and nonpathogenic bacteria were studied in this paper. Hemocytes of H. dicersicolor were classified by their characteristics of morphology and immune function. The process of hemocytes phagocytosis to bacteria was also researched. Different defensive mechanism of cellular and humoral immunity of abalones toward pathogenic vibrios and nonpathogenic Escherichia coli, as well as their extracellular products (ECP), was discussed too. Effects of antibiotics on immune function of abalone’s hemocytes were also expatiated. There were 3 parts of results in this paper.Part 1: Observations of morphology and immune function of hemocytes in H. dicersicolor.Mortalities and agglutinabilities of hemocytes were as indexes for the first time to screen feasible anticoagulants of gastropods among 3 ones confected in our labs i.e. HA1, HA2, RH and 6 ones from other invertebrates. It was showed that in HA1 and RH, abalone’s hemocytes were equably distributing and mortalities of hemocytes were lower than 6% in 6h and 33% in 24h. As a result, HA1 and RH were the best anticoagulants of abalone’s hemolymph.According to their structural characteristic accompanied with karyoplasmic ratios and immune function, hemocytes of H. dicersicolor were firstly classified into 3 types. The first type was of sizes 7.1~8.6μm×7.8~9.2μm and karyoplasmic ratio 1.39. There were many bacillary, elliptical or oval hollow granules and a few mitochondria in their cytoplasm. They could phagocytize bacteria and yeasts and not reduce nitroblue tetrazolium (NBT). The type of hemoctes was thus called as granulocytes (Type I). The other two types were agranulocytes because there were no granules in their cytoplasm. The size of the bigger one was 6.7~8.1μm×7.1~8.3μm with the karyoplasmic ratio of 1.82. Their cytoplasms were rich in endoplasmic reticulums. They could phagocytize bacteria, adhere to yeasts and also reduce NBT. This type of the bigger agranulocytes was entitled hyalinocytes (Type II) based on the above characteristics. The smaller aganulocytes were of sizes 4.56～6.49μm×3.82～5.75μm and karyoplasmic ratio 3.24. There were more mitochondria in their cytoplasm than in the other two types. They had no phagocytic activities and couldn’t reduce NBT. They were therefore identified as like-lymphoid cells (TypeⅢ).Part 2: Studies of immune function and its influential factors of hemocytes in H. dicersicolor in vitro.In gastropod immunology, effects of flocculations of pathogenic vibrio (F3602 and X4302) and nonpathogenic Escherichia coli on immune function of hemocytes in vitro, including chemotaxis, products of reactive oxygen species (ROS), phagocytic activities and integrality of lysosomal membranes evaluated by the neutral red retention (NRR) assay, were reported for the first time. It was showed that at the same concentration, flocculations of 2 vibrios leaded to abalones’hemocytes producing insignificantly less ROS and destroying lysosomal membranes more badly than those of E. coli (P<0.05). Hemocyte’s chemotactic distances in flocculations of F3602 and X4302 (2.1×10~7cfu/mL) were 1.69 (P <0.05) and 1.57 times in that of E. coli. At the concentration of 6.3×10~7cfu/mL, hemocytes phagocytized 2 vibrios significantly more than E. coli (P<0.05).Actions of bacterial ECP on the above immune function of gastropod’s hemocytes in vitro were firstly researched. When protein concentrations of 2 vibrio ECPs were 30mg/L, they destructed lysosomal membranes more severely than E. coli ECP (P<0.05). At the low protein concentration(7.5mg/L), hemocytes’chemotaxis by X4302和F3602 ECPs was obviously larger than that by E. coli ECP (P<0.05). At the same protein concentration, hemocytes produced insignificantly more ROS in ECPs of 2 vibrios than those in E.coli ECP. Added by high protein concentration F3602 ECP (30mg/L), phagocytic activities of hemocytes were markedly higher than that by E.coli ECP (P<0.05).It was firstly reported that antibiotics could affect immune function of shellfish’s hemocytes in vitro. Neomycin, spectinomycin and streptomycin, which belonged to aminoglycoside antibiotics, as well as chloramphenicol were 4 selected antibiotics which could inhibit vibrios F3602 and X4302 from reproducing as part 2. As a result, the effect of streptomycin on immune function of abalone’s hemocytes was sharply different from the other three ones. Compared with the control (added by the normal saline), when added by streptomycin solutions, haemocytes produced insignificantly more ROS (P>0.3). Phagocytic activities of hemocytes were markedly increased (P<0.05). Membranes of lysosome were not destroyed severely and chemotaxis of hemocytes was inhibited lightly. So streptomycin may be a good drug to cure vibriosis of H. dicersicolor. Hemocytes phagocytized less F3602 in neomycin solution than that in streptomycin one at the same concentration. When concentrations of neomycin were greater than 50μg/mL, it sharply decreased hemocytes’chemotaxis (P<0.05). Accordingly neomycin was not fit for curing abalones’vibriosis. Chloramphenicol and spectinomycin did not promote phagocytic activities of haemocytes as greatly as spectinomycin, severely destroyed lysosomal membranes (P<0.05), consumingly restrained haemocytes from producing ROS when their concentration was 100μg/mL (P<0.05) and evidently reduced chemotactic distances of abalone’s haemocytes at concentrations of 100μg/mL and 50μg/mL (P<0.05). Consequently, these 2 antibiotics would be better to be forbidden applying in abalone culture.At different temperature, the same factors could affect immune function of abalones’hemocytes unlikely. Comparison with at 30℃, F3602 flocculation and its ECP induced hemocytes to increase phagocytic activities and decrease products of ROS and chemotaxis and lightly destroy lososomal membranes at 15℃. While in the 15℃antibiotic experiment, products of ROS of abalones’hemocytes were lightly fluctuated, phagocytic activities were appreciably decreased and destruction of lysosomal membranes was lightened compared with in the 30℃one. Except for groups added by neomycin and spectinomycin (100μg/mL), chemotactic distances of hemocytes were shortened at 15℃. In conclusion, low temperature could lighten destruction effects of pathogens and their ECP on lysosomal membranes of abalones’hemocytes and be benefit for immune systems of shellfish.Part 3: Effects of infections by vibrios and E. coli and their ECP on immune function of hemocytes in vivo, as well as activities of 3 enzymes and contents of like-Immunoglobulins in sera of H. dicersicolor In artificial infections, vibrio F3602 and its ECP were lethal to healthy H. dicersicolor. Lethal Dose 50% (LD50) of F3602 and its ECP in 48h and 96h were respectively 6.88×10~7cfu/mL and 2.84×10~7cfu/mL, 252.9mg/L and 183.2mg/L. After injected by flocculations of F3602 and E.coli and their ECP at the concentration below 48h LD50, changes of immune function of hemocytes and humoral factors in sera of H. dicersicolor were studied. Compared with the control injected by the normal saline, when injected concentrations of F3602 were higher than 8.6×10~6cfu/mL, lysosomal membranes of hemocytes were markedly destructed (P<0.05) and chemotaxis was sharply inhibited (P<0.05) as well as products of ROS of hemocytes were significantly increased (P<0.05). While injected concentrations of F3602 were 8.7×106cfu/mL and 5.8×10~6cfu/mL, phagocytic activities of hemocytes were obviously increased (P<0.05). Hemocytes of abalones injected by F3602 ECP, whose protein concentration were 204mg/L, produced less ROS (P<0.05) and phagocytized less F3602 (P<0.05) than those injected by the normal saline. At the same time, their lysosomal membranes were more badly destroyed (P<0.01) and chemotactic distances were longer (P<0.05). The above 4 items of immune function of abalones’hemocytes hardly changed after they were injected by E. coli and its ECP at low concentration. However, infected by E. coli flocculation of the high concentration (5.22×10~7cfu/mL), phagocytic activities were obviously hastened (P<0.05) and lysosomal membranes were extremely significantly destructed (P<0.01). E. coli ECP of high protein concentration (204mg/L) only extremely increased products of ROS compared with the control group (P<0.01).Having injected for 48h, changes of relative enzyme activities of alkaline phosphatase (ALP), acid phosphatase (ACP) and superoxide dismutase (SOD) in sera of H. dicersicolor infected by flocculations of F3602 and E. coli were analysed and synchronously effects of injections by bacterial ECP on activities of the 3 enzymes in shellfish’sera were reported in shellfish immunology for the first time. It was showed that compared with the control, relative activities of SOD in sera of abalones were significantly inhibited after abalones were infected by high concentrations F3602 (5.22×10~7cfu/mL) and F3602 ECP solutions (whose protein concentrations were 204mg/L) (P<0.05); low concentration E.coli (8.7×10~6cfu/mL) and high concentration F3602 flocculations could markedly increased relative activities of ALP in sera; Relative activities of ACP were sharply increased because H. dicersicolor were injected by low concentration E.coli flocculation and F3602 ECP (whose protein concentrations were 204mg/L) (P<0.05) and obviously decreased because those by F3602 ECP at high protein concentration (P<0.05).It was firstly reported that in sera of gastropods were there immunoglobulins like proteins (like-Igs) that were similar with human IgA, IgG and IgM and could react specific responses to human antisera. Contents of like-IgA, like-IgG and like-IgM were 64～71μg per milliliter sera of H. dicersicolor before infected by bacteria. After induced by E. coli (8.7×10~6cfu/mL), contents of like-IgA, like-IgG and like-IgM were greatly increased to 157.008±18.201μg/mL, 100.252±6.870μg/mL, 88.019±6.599μg/mL respectively. Contents of like-IgM in sera of abalones were the same order of magnitude as other animals and human beings, while like-IgA, like-IgG were 1 or 2 order of magnitude lower than other animals.Actions of vibrio F3602 flocculation and its ECP on contents of like-Igs in sera of shellfish were compared with those of nonpathogenic E. coli and its ECP for the first time. Whether injected by F3602 flocculation of low concentration (8.7×10~6cfu/mL) and its ECP or by E. coli and its ECP at the same concentration, contents of like-IgA in sera of H. dicersicolor were significantly higher than the control group injected by the normal saline (P<0.05); Similarly, contents of like-IgG were also sharply increased when abalones were injected by F3602 flocculation and its ECP of low concentration (P<0.05). Nevertheless, induced actions of F3602 and its ECP on contents of like-IgM were different from E. coli and its ECP. It was incorporated that when injected by F3602 of low concentrations, contents of like-IgM were markedly lower than that by E. coli at the same concentration (P<0.05). By contrary, when injected by F3602 of high concentrations, its contents were significantly higher than that by E. coli (P<0.05). Compared with the control, injected by F3602 ECP (whose protein concentrations were 204mg/L and 51mg/L) as well as E. coli ECP of low protein concentrations (51mg/L), contents of like-IgM were obviously decreased (P<0.05), extremely significantly (P<0.01) and markedly (P<0.05) increased respectively.更多还原