致对虾肝胰腺坏死病副溶血弧菌荧光定量PCR检测方法的建立

【目的】建立一种对虾急性肝胰腺坏死病（Acute Hepatopancreatic Necrosis Disease）病原菌副溶血弧菌（Vibrio parahaemolyticus, VpAHPND）荧光定量 PCR 检测方法,对该病进行快速检测。【方法】根据副溶血弧菌基因保守序列设计特异性引物和探针,经优化反应体系及条件,建立检测 VpAHPND 的荧光定量 PCR 方法;以重组质粒为标准品制作标准曲线,并进行特异性、敏感性、重复性及临床应用试验。【结果】该方法定量范围宽,起始模板浓度范围为 2.3×101 ~2.3×107 拷贝 /μL 时,标准曲线具有良好的线性关系;最低检测限为 2.3 拷贝/μL,与对虾其他病原菌及病毒无交叉反应,重复试验变异系数为 0.45%~0.69%,可在 1 h 内完成样品检测;对临床对虾样品的检测结果表明,该方法与世界动物卫生组织（OIE）推荐的套式 PCR 法检测结果一致。【结论】应用 TaqMan 探针建立的检测 VpAHPND 的荧光定量 PCR 方法具有灵敏度高、特异性强、重复性好的特点,可有效用于临床对虾样品的 VpAHPND 检测。     

灌喂必需氨基酸模式溶液对草鱼全鱼_省略_肌肉_肝胰脏蛋白质合成代谢的影响

采用大剂量食道灌喂DL－[4-^3H]-Phe，研究不同EAA模式下，20－30g体重草鱼种的全鱼和肌肉，肝胰脏的蛋白质合成代谢，结果表明：（1）分别缺乏Lys,Met,Trp Arg的4种极端不平衡EAA模式，同草鱼肌肉EAA模式即相对平衡的基础模式相比，肌肉，肝胰脏及全鱼的蛋白质合成速率（FSR）均显著降低，其降低程度高低依次为Met,Lys,Arg,Trp;(2)肝胰脏，肌肉人的蛋白质合成代谢与整体蛋白质合成代谢对EAA模式的改变产生相同的变化趋势，并且三者蛋白质合成代谢速率为肝胰脏＞全鱼＞肌肉；（3）EAA模式的Lys,Met,Trp,Arg4因素中，对肌肉，肝胰脏蛋白质合成速率影响最大的是Met,其余依次为Lys,Arg,Trp。     

Haemolymph metabolic variables in relation to eyestalk ablation and gonad development of Pacific white shrimp Litopenaeus vannamei Boone

The present study analyses some biochemical variables in haemolymph proposed as predictive indicators of the maturation capability following eyestalk ablation. Haemolymph of captive females was obtained before and 8 days after eyestalk ablation, and levels of haemocyanin, total proteins, glucose, lactate, cholesterol, triacylglycerides and vitellogenin were determined. Biochemical variables were also analysed in the hepatopancreas at the end of the experiment. Females were grouped as immature (previtellogenic stage) and mature (vitellogenic and cortical stages) based on histological analysis done 8 days after eyestalk ablation. To analyse haemolymph variables before eyestalk ablation in relation to maturation capability, immature females were classified as those with a low maturation capability and mature females as those with a high maturation capability. Females of high maturation capability had significantly higher vitellogenin levels before eyestalk ablation than females of low maturation capability. No significant differences were found for the other biochemical variables. Vitellogenin was also higher in mature than in immature females at the end of the experiment. These results indicate that vitellogenin levels in haemolymph could be used as possible predictive criteria of maturation capability, possibly because they reflect the degree of ovarian development at the time of eyestalk ablation.     

中华绒螯蟹蜕皮过程中肌肉、肝胰脏和甲壳中钙和磷含量的变动

对中华绒螯蟹蜕皮间期和蜕皮后24h、48h和96h肌肉、肝胰脏及甲壳中钙和磷的含量进行了测定。蜕皮后肌肉中钙的含量较间期低，但从24h到48h有所增加。肝胰脏中钙的含量在蜕皮后各时间内无显著变化，甲壳中钙的含量在蜕皮后24h到96h呈增加趋势。甲壳的矿化作用在蜕皮后96h已接近完成。蜕皮后肌肉中磷的含量较间期高，以后变化不明显。蜕皮后肝胰脏中磷的含量高于间期，甲壳中含量较间期低。蜕皮后肌肉中钙磷比迅速降低，肝胰脏中钙磷比在蜕皮后24h无明显变化，但在蜕皮后24h至96h迅速降低。中华绒螯蟹蜕皮后外表皮对钙的矿化速度显著快于磷的矿化速度。     

Presence and ontogeny of intestinal and pancreatic phospholipase A2-like proteins in the Red Sea bream,Pagrus major. An immunocytochemical study

We have studied the location and the ontogeny of the digestive enzyme, phospholipase A₂ (PLA₂) immunohistochemically in the adult and larvae/juvenile of the red sea breamPagrus major by using an antiserum against theNaja naja venom PLA₂. The antiserum reacts with at least one enzyme among the PLA₂s purified from the fish hepatopancreas or intestine. Although the reactivities were comparatively low, it labelled zymogen granules of the pancreatic acinar cells and secretory materials of certain epithelial cells in the depths of epithelial crypts in the pyloric caeca of the adult. The immunoreactivities of PLA₂s were investigated in the viscera of larvae and juveniles of the 0 to 85^th day after hatch. In the larvae of the 13^th day, accumulation of PLA₂-positive zymogen granules in the pancreatic acinar cells were first recognized by the immunostaining. The intensity of the labelling subsequently became stronger and dramatically increased between the 20^th and 30^th day. This increase appeared to be one of the physiological changes associated with the transition to a new benthic life as juveniles. Lack of PLA₂ in the pancreas before the 13^th day may suggest the possibility that larvae utilized exogenous PLA₂, inherent in their prey, to digest the phospholipids. On the other hand, no reactivity was found in the intestine until the 85^th day.     

中华绒螯蟹血淋巴和肝胰腺的总蛋白含量与性早熟的关系

中华绒螯蟹扣蟹性腺发育与肝胰腺和血淋巴的蛋白质浓度相关.性早熟前后,雌雄扣蟹肝胰腺总蛋白浓度分别由(121.17±44.50)mg@g-1和(89.43±21.86)mg@g-1下降至(81.08±5.84)mg@g-1和(70.58±9.90)mg@g-1,雌性扣蟹的肝胰腺占体重的比值也由8.18%降至3.90%.血淋巴总蛋白浓度正常发育的雌性扣蟹为(99.55±12.06)mg@mL-1,明显高于性早熟雌性扣蟹的(72.26±17.87)mg@mL-1;而雄性扣蟹性早熟前后的血淋巴总蛋白浓度无显著差异.肝胰腺总蛋白浓度二秋龄雌蟹高于性早熟雌性扣蟹,但它们之间的血淋巴总蛋白浓度没有显著差异.二秋龄雄蟹的血淋巴和肝胰腺总蛋白浓度与性早熟雄性扣蟹相当,差异不显著.     

Cu2+对克氏原螯虾（Procambarus clarkii）肝胰腺的蛋白质氧化损伤效应

克氏原螯虾(Procambarus clarkii)是一种潜在的水质环境污染的指示生物,为研究水体中不同浓度的重金属Cu2+对其肝胰腺组织的蛋白质氧化损伤作用,在测得Cu2+致克氏原螯虾96 hLC50为30 mg·L-1后,将螯虾分别暴露于0、0.5、1.0、3.0、5.0、10.0 mg·L-1的Cu2+溶液中,并在暴露24、48、72、96 h后测定各处理组肝胰腺组织的活性氧自由基(ROS)、总抗氧化能力(T-AOC)、谷胱甘肽过氧化物酶(GSH-PX)活性、蛋白质羰基(PCO)含量以及DNA-蛋白质交联(DPC)系数。结果表明,在暴露24h时,随着Cu2+浓度的升高,ROS、T-AOC和GSH-PX水平的变化规律相似,均为先上升后下降,PCO含量和DPC系数随暴露浓度的升高而增加;在暴露48、72、96 h后,ROS、T-AOC、PCO水平和DPC系数均表现为先升后降的趋势,GSH-PX活性在染毒96 h后,随着浓度的升高而下降。PCO含量的变化具有一定的剂量-效应关系。结果提示,肝胰腺的蛋白质氧化损伤可能是重金属Cu2+致克氏原螯虾毒性效应的主要机制之一。     

草鱼幼鱼肝胰脏抗氧化系统对MC-LR胁迫的响应

为了研究微囊藻毒素(MC-LR)对草鱼(Ctenopharygodon idella)幼鱼肝胰脏抗氧化防御系统的影响,本研究通过腹腔注射的方法(剂量为25、100μg MC-LR·kg-1,分别称为低剂量和高剂量),对草鱼幼鱼进行染毒,于胁迫24、48 h和72 h后分离其肝胰脏,随后采用分光光度法检测了草鱼幼鱼肝胰脏中超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性;同时采用荧光定量PCR方法分析了SOD、CAT、谷胱甘肽过氧化物酶(GPx)和谷胱甘肽还原酶(GR)基因相对表达量的变化。结果显示:高剂量MC-LR诱导24 h和48 h后,草鱼幼鱼肝胰脏SOD活性显著增加(P0.05),而SOD在低剂量组中活性没有显著变化(P0.05);低剂量MC-LR对CAT活性没有显著影响(P0.05),而高剂量MC-LR诱导24 h后可使草鱼幼鱼肝胰脏CAT活性显著增加(P0.05),随后其活性又下降,但差异不显著(P0.05)。在MC-LR胁迫过程中,SOD、CAT、GPx基因表达在两个剂量组中均显著低于对照组(P0.05),而GR基因在低剂量MC-LR胁迫24 h后相对表达量显著上调(P0.05),尽管在72 h相对表达量上调,但差异不显著(P0.05),而高剂量组中GR基因在胁迫24 h和48 h后,其表达量被抑制,但不存在显著差异(P0.05)。进行抗氧化酶活性和基因表达相关性分析发现,SOD和CAT酶活性与SOD和CAT基因表达不相关。上述研究表明MC-LR对草鱼幼鱼肝胰脏抗氧化系统产生了明显的胁迫效应,但MC-LR对机体抗氧化酶活性与基因编码调控之间的相互作用机制还有待更深入的研究。     

日本新糠虾卵巢和肝胰腺主要细胞内5-HT的免疫组织化学定位

运用免疫组化方法,观察了5-HT阳性物质在日本新糠虾不同发育期的卵巢卵细胞和滤泡细胞及肝胰腺细胞中的分布和变化。结果表明:随卵细胞的发育,滤泡细胞逐渐迁移至其近旁。各期卵巢卵细胞和滤泡细胞及肝胰腺细胞中均存在大量5-HT阳性细胞,阳性物质呈深棕色;各期卵巢中5-HT在滤泡细胞内均呈阳性;在Ⅰ、Ⅱ和Ⅲ期卵巢中卵细胞胞质和胞核均呈阳性,且阳性强度呈递减;Ⅳ期卵巢中5-HT在卵细胞中呈阴性,而在Ⅴ期卵巢中5-HT在卵细胞中又呈现弱阳性。5-HT在发育各期的肝胰腺细胞中均呈阳性。同一期卵巢中,不同类型肝胰腺细胞间5-HT的分布没有差别。除Ⅳ期卵巢外,在其余各期卵巢中5-HT阳性强度在卵细胞胞质和肝胰腺细胞胞质中表现一致。