扇贝(Chlamys farreri)多肽通过EGFR抑制受紫外线B(UVB)诱导的HaCaT细胞凋亡

利用Hoechst 33258荧光染色法检测紫外线B(UVB)辐射诱导HaCaT细胞凋亡率。结果表明,扇贝多肽(Polypeptide from Chlamys farreri,PCF)可以剂量依赖性抑制UVB诱导的HaCaT细胞凋亡;表皮生长因子受体(EGFR)抑制剂AG1478能明显抑制UVB诱导的HaCaT细胞凋亡。采用3’-RACE法构建EGFR的cDNA片段,克隆测序检测突变位点。结果表明,UVB照射后EGFR发生碱基突变A→G,A→G,T→C,G→A,G→A;预加入5.69mmol/L的PCF,产生部分抗突变作用,1,2,3突变位点处未发生碱基的突变。     

Characterization,expression and function analysis of DAX1 gene of scallop (Chlamys farreri jones and preston 1904) during its gametogenesis

DAX1, a member of nuclear receptor superfamily, has a function in the sex determination and gonadal differentiation of several vertebrate species. However, little information about DAX1 of invertebrates is available. Here we cloned a homolog of scallop (Chlamys farreri Jones and Preston 1904) dax1, Cf-dax1, and determined its expression characteristics at mRNA and protein levels. The cDNA sequence of Cf-dax1 was 2093 bp in length, including 1404 bp open reading frame (ORF) encoding 467 amino acids. Unlike those of vertebrates, no conserved LXXLL-related motif was found in the putative DNA binding region of Cf-DAX1. Fluorescence in situ hybridization showed that Cf-dax1 located on the short arm of a pair of subtelocentric chromosomes. Tissue distribution analysis using semi-quantitative RT-PCR revealed that Cf-dax1 expressed widely in adult scallop tissues, with the highest expression level found in adductor muscle, moderate level in mantle, gill and testis, and low level in kidney, ovary and hepatopancreas. The result of quantitative real-time PCR indicated that the expression of Cf-dax1 was significantly higher (P<0.05) in testis than in ovary at the same stage, showing a sex-dimorphic expression pattern. Furthermore, immunohistochemical detection found that Cf-DAX1 mainly located in spermatogonia and spermatocytes of testis and in oogonia and oocytes of ovary, implying that DAX1 may involve in gametogenesis of bivalves.     

栉孔扇贝对氮杂螺环酸毒素代谢解毒的生理应激响应

将栉孔扇贝（Chlamys farreri）暴露于分离自我国近海的一株氮杂螺环酸毒素（azaspiracids，AZAs）产毒藻——腹孔环胺藻（Azadinium poporum，AZDY06株，主产AZA2），研究了栉孔扇贝对AZAs的代谢解毒效应以及代谢过程中血细胞数量、抗氧化酶活性及细胞超微结构变化等生理应急响应。结果发现，栉孔扇贝对AZDY06所产的AZA2具有一定的代谢解毒能力，可将AZA2转化成AZA6、AZA12、AZA19和AZA23等4种极性较强且毒性较低的代谢产物。代谢解毒过程中，栉孔扇贝血细胞数量、关键抗氧化酶活性及靶器官细胞超微结构均出现一定的应激效应，且总体表现出明显的剂量-效应关系。栉孔扇贝血细胞数量对AZAs表现出较强的应激反应，且随实验过程出现较大波动，最高可达对照组的2—3倍；抗氧化系统酶如超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）、过氧化物酶（POD）和酸性磷酸酶（ACP）活性也出现不同程度的变化：GSH-Px与POD在整个蓄积代谢过程中均表现出持续激活状态，且血液中这两种酶活性最高，可达内脏和鳃中的20倍以上；SOD活性与扇贝中AZAs含量有一定关系，AZAs含量为74.2—168.2μg AZA1 eq/kg时为诱导状态，过高或过低剂量均表现为抑制效果；而ACP对AZAs的应激表现也十分敏感，但代谢过程中大部分时间为被持续抑制状态。此外，AZAs还导致栉孔扇贝内脏和鳃细胞超微结构出现空泡化、核固缩等病理性变化；实验后期，低暴露组细胞结构出现一定的自我修复现象，而高暴露组则无此现象。本研究可为进一步深入探索AZAs的解毒机制，对其进行风险评价并制定限量标准提供科学依据。     

原油污染对栉孔扇贝抗氧化酶活性的影响

以原油水溶性成分(water soluble fraction of crude oil,WSF)为污染物,采用暴露实验法,研究了栉孔扇贝(Chlamys farreri)鳃和消化腺组织中超氧化物歧化酶(Superoxide Dismutase,SOD)和过氧化氢酶(Catalase,CAT)活性的变化.结果表明,WSF污染下,鳃和消化腺组织SOD和CAT酶活性随暴露时间增加一般表现为降低-升高-降低的趋势,酶活性达到最高的时间随浓度不同而变化.第1天时消化腺SOD在0.08 mg/L浓度下被诱导,而后随时间增加表现为抑制效应;浓度为0.21和0.88 mg/L时消化腺SOD酶活性被抑制,随暴露时间延长而活性增加.暴露时间为4d时,石油烃浓度在0.08和0.88 mg/L时鳃组织SOD酶活性均被抑制,而浓度为0.21 mg/L时被诱导.消化腺和鳃组织SOD可以作为扇贝被污染胁迫的指标.     

栉孔扇贝(Chlamys farreri)壳顶幼虫血细胞分布的免疫学观察

研究组前期实验发现,栉孔扇贝血细胞在D形幼虫时期出现.本文应用单克隆抗体的免疫组化、免疫电镜方法进一步定位栉孔扇贝壳顶幼虫的血细胞并观察其分布.结果表明,栉孔扇贝壳顶幼虫已分化出界限明显的组织器官,包括外套膜、面盘、口、食道、胃、消化腺、足等;血细胞主要分布于壳顶幼虫的外套膜、面盘、食道、消化腺、胃等组织器官内及周围,其中面盘、消化腺、胃等处有大量血细胞成簇分布.血细胞形状不规则,直径为3～5μm;细胞核多为圆形、椭圆形,位于细胞的一侧;细胞质内有线粒体、内质网等细胞器和空泡.结果证实,栉孔扇贝在壳顶幼虫时期已出现了大量清晰可辨的血细胞,其分布特点与成贝血细胞相似.     

Isolation and Biochemical Characteristics Analyses of Phenoloxidases (POs) in Three Cultured Mollusk Species

Phenoloxidases(POs)are a group of copper proteins including tyrosinase,catecholase and laccase,which play crucial roles in the innate immune response of mollusks.In this research,POs were studied in cultured mollusk species,including scallop Chla-mys farreri,abalone Haliotis discus hannai and clam Scapharca subcrenata.The POs were isolated from hemocytes using linear-gradient native-PAGE combined with catechol staining.The PO activities and their characters were investigated.The molecular mass of PO in C.farreri was 576 kDa,and it was 228 kDa in H.discus hannai.In S.subcrenata,four POs were detected and their mole-cular masses were 391 kDa,206 kDa,174 kDa and<67 kDa,which were named as 391-PO,206-PO,174-PO and s-PO,respectively.Ki-netic analyses indicated that all of the POs,except for 391-PO had higher affinity to L-DOPA and catechol than to hydroquinone and dopamine.However,all of the POs failed to oxidize tyrosine.The effects of divalent metal ions on POs’activities were assayed,in-cluding Fe²⁺,Mg²⁺,Zn²⁺,Mn²⁺,Cu²⁺and Ca²⁺from FeCl₂,MgSO₄,ZnSO₄,MnCl₂,CuSO₄and CaCl₂.The POs were inhibited by Fe²⁺at all determined concentrations.Additionally,the inhibition assay showed that all of the POs were inhibited by cysteine,ascorbic acid,sodium sulfite,citric acid,ethylenediaminetetraacetic acid disodium(EDTA)and sodium diethyldithiocarbamate(DETC).The inhibition effects of critric acid and EDTA are dose-dependent.H.discus hannai PO and 391-PO were slightly inhibited by sodium azide,and H.discus hannai PO,391-PO and 174-PO were slightly inhibited by thiourea.In conclusion,the POs in the three cultured mollusks are copper-containing laccase-type phenoloxidases with similar biochemical characteristics even though their molecular masses are different.     

缩短栉孔扇贝养殖周期新措施的探讨

本文主要介绍了利用栉孔扇贝三倍体，升温苗的养成及强化养成管理等新措施来达到缩短栉孔扇贝养殖周期，并取得了较好的效果。     

一种栉孔扇贝寄生虫——车轮虫的初步研究

2000年夏季，对在胶南、蓬莱、烟台三个养殖区采集到的发生大面积死亡前后的栉孔扇贝进行组织学研究，发现鳃丝和外套腔内寄生有大量的车轮虫，并伴随有大量的黏液产生。车轮虫和黏液的平均检出率分别为18％和12％。本对此类车轮虫进行形态学、病理学等方面的研究，并指出了其对栉孔扇贝的危害作用。     

栉孔扇贝血细胞的显微和超微结构

通过光镜、扫描电镜和透射电镜观察栉孔扇贝Chlamys farreri血细胞的形态、结构特征,对其分类进行了初步研究。根据细胞的大小、形态和结构特征可将血细胞分成5种类型:Ⅰ型小透明细胞,大小约2.44±0.11μm,约占45%—50%;Ⅱ型大透明细胞,大小约4.83±0.28μm,约占15%—20%;Ⅲ型小颗粒细胞,大小约4.07±0.15μm,约占15%—20%;Ⅳ型中颗粒细胞,大小约7.20±0.26μm,约占20%—25%和Ⅴ型大颗粒细胞,大小约13.87±0.73μm,约占3%—5%。血细胞在血淋巴中的平均密度为(3.03±0.11)×107cell.ml—1,其中颗粒细胞占42.6%,透明细胞占57.4%。透射电镜观察结果表明,颗粒细胞的细胞质颗粒可区分成3种类型:Ⅰ型高电子密度颗粒,Ⅱ型低电子密度颗粒和Ⅲ型中等电子密度颗粒。     

桑沟湾养殖栉孔扇贝食物来源研究——脂肪酸标志法

运用脂肪酸标志法分析了桑沟湾养殖栉孔扇贝(Chlamys farreri)春、夏季食物来源组成。结果表明,桑沟湾养殖栉孔扇贝饵料组成中含有硅藻类、细菌、大型绿藻类、鞭毛藻类等饵料来源,且各种饵料来源比例具有较显著的季节变化。采样期间硅藻脂肪酸标志16:1/16:0(相对质量分数比,下同)从0.91降至0.45,表明扇贝硅藻类饵料来源比例呈逐渐下降趋势;细菌脂肪酸标志18:1n-7/18:1n-9比值从0.31升至0.90,奇数碳、支链脂肪酸相对质量分数从2.15%升至3.28%,表明扇贝细菌类饵料来源比例呈逐渐上升趋势;绿藻脂肪酸标志Σ(18:2n-6 18:3n-3)质量分数从2.78%升至5.77%,表明大型绿藻来源有机质饵料比例逐渐上升。栉孔扇贝脂肪酸组成中较高比例的DHA表明扇贝摄取了富含DHA的有机质,如鞭毛藻类、微型浮游动物等。栉孔扇贝食物来源的季节变化很可能是由水体中潜在食物来源的季节变化所导致。