菲律宾蛤仔糖胺聚糖抗肿瘤活性研究

从菲律宾蛤仔全脏器中提取得到糖胺聚糖粗品(CRG),通过DEAE-52离子交换柱层析得到纯品(RG),分别采用MTT法和动物移植性肿瘤模型检测其体外和体内抗肿瘤活性,同时测量荷瘤小鼠的胸腺指数的脾指数;建立小鼠免疫抑制模型,并测量正常小鼠和免疫抑制小鼠的胸腺指数、脾指数和碳粒廓清指数。结果显示,菲律宾蛤仔糖胺聚糖可以抑制体外培养的人肝癌Bel7402肿瘤细胞的生长,能显著抑制移植性S180肉瘤的生长,可抑制荷瘤小鼠的脾肿大现象,对环磷酰胺造成的小鼠胸腺和脾脏萎缩有抵抗和修复作用。     

Antitumor and immune regulation activities of the extracts of some Chinese marine invertebrates

Extracts of 21 marine invertebrates belonging to Coelenterata, Mollusca, Annelida, Bryozoa,Echiura, Arthropoda, Echinodermata and Urochordata were screened for the studies on their antitumor and immune regulation activities. Antitumor activity was determined by MTT method and immune regulation activity was studied using T- and B-lymphocytes in mice spleen in vitro. It was found that the n-butanol part of Asterina pectinifera, the acetic ether part of Tubuaria marina, 95% ethanol extract of Acanthochiton rubrolineatus have a high inhibition rate of 96.7%, 63.9% and 50.5% respectively on tumor cell line HL-60 at the concentration of 0.063 mg/ml. The inhibition rate of the acetic ether part of Tubuaria marina on the tumor cell line A-549 is 65,4 % at concentration of 0.063 mg/mL. The 95% ethanol extract of Meretrix meretrix has so outstanding promoting effect on T-lymphocytes that their multiplication increases 25% when the sample concentration is only 1μg/ml. On B-lymphocytes, the 95% extract of Rapana venosa, at concentration of 100μg/ml, has a promotion percentage of 60%. On the other hand, under the condition of no cytotoxic effect, the 95% ethanol extracts of Acanthochiton rubrolineatus and Cellana toreum can reach 92% inhibition rate on T lymphocyte at concentration of 100μg/ml, while the inhibition rate on B lymphocyte of the 95% extract of Acanthochiton rubrolineatus reaches 92% at the same concentration.     

生物絮团对养殖环境的清洁作用及对虾生理指标的影响

本文研究了生物絮团对养殖环境的清洁作用及对虾生理指标的影响。生物絮团实验梯度设置为0、5、10 ml/L,在实验开始后将凡纳滨对虾随机放入氨氮浓度(2.5±0.3)mg/L的实验水槽中,以不添加生物絮团组为对照组,分别于0,6,12,24,48h时取对虾血淋巴,鳃丝、肝胰脏样品并进行免疫和解毒代谢指标测定。结果表明:生物絮团能有效地吸收水中的氨氮,来提高虾体免疫活性和解毒代谢能力。各处理组水中氨氦随着时间的推移逐渐降低,24h之后维持在一个较低水平。各处理组虾体血液、肝胰脏、鳃丝中精氨酸酶活力、谷氨酰胺合成酶活力随着生物絮团将水体中氨氮的清除逐渐降低,在24h,凡纳滨对虾鳃丝、血淋巴、肝胰脏中谷氨酰胺合成酶在生物絮团处理组之间以及处理组和对照组相比均差异显著(P0.05),并且其活性下降程度与生物絮团浓度成正比。各处理组对虾血细胞数量,吞噬率,溶菌,抗菌活力在施加生物絮团后呈现先上升,后下降趋势,48h降低在一个较低水平。由此可见,生物絮团对养殖水体中氨氦有明显的清洁作用,并对凡纳滨对虾免疫指标和解毒代谢指标影响显著。     

中国明对虾Imd免疫信号通路相关基因克隆及表达分析

Imd免疫信号通路在无脊椎动物抗革兰氏阴性菌方面起到十分重要的作用。在已知中国明对虾Imd通路下游核转录因子Relish基因全长的情况下,利用同源克隆方法获得了Imd通路上游基因Imd的cDNA序列,命名为FcImd,该基因全长783bp,5’非编码区45bp,3’非编码区255bp,开放阅读框483bp,编码160个氨基酸,包括一个位于C端的死亡结构域。同源性分析表明,FcImd基因氨基酸序列与其他甲壳动物的氨基酸序列高度保守,与凡纳滨对虾和日本囊对虾的同源性分别为99%和88%。于鳗弧菌感染后的1,3,6,12,24,48,96,144h记录实验组和对照组的累积死亡率,并分别取血淋巴用于检测中国明对虾Imd及Relish基因在不同时间点的表达量变化情况。结果显示:鳗弧菌感染后,实验组Imd和Relish表达量均显著高于对照组(P0.05),其中Imd表达量呈先升高后降低趋势,Relish表达量在1h时达到最高,然后呈逐渐降低趋势。中国明对虾感染鳗弧菌后Imd和Relish基因呈不同步上调表达,说明Imd和Relish基因的表达与弧菌刺激密切相关,提示Imd和Relish基因参与了对虾抗菌反应的Imd信号转导通路。     

Effects of Pseudoalteromonas sp.BC228 on Digestive Enzyme Activity and Immune Response of Juvenile Sea Cucumber （Apostichopus japonicus）

A marine bacterium,Pseudoalteromonas sp.BC228 was supplemented to feed in a feeding experiment aiming to determine its ability of enhancing the digestive enzyme activity and immune response of juvenile Apostichopusjaponicus.Sea cucumber individuals were fed with the diets containing 0 （control）,105,107 and 109 CFU g-1 diet of BC228 for 45 days.Results showed that intestinal trypsin and lipase activities were significantly enhanced by 107 and 109 CFU g-1 diet of BC228 in comparison with control （P〈0.01）.The phagocytic activity in the coelomocytes of sea cucumber fed the diet supplemented with 107 CFUg-1 diet of BC228 was significantly higher than that of those fed control diet （P〈0.05）.In addition,105 and 107 CFUg-1 diet of BC228 significantly enhanced lysozyme and phenoloxidase activities in the coelomic fluid of sea cucumber,respectively,in comparison with other diets （P 〈 0.01）.Sea cucumbers,10 each diet,were challenged with Vibrio splendidus NB 13 after 45 days of feeding.It was found that the cumulative incidence and mortality of sea cucumber fed with BC228 containing diets were lower than those of animals fed control diet.Our findings evidenced that BC228 supplemented in diets improved the digestive enzyme activity of juvenile sea cucumber,stimulated its immune response and enhanced its resistance to the infection of V.splendidus.     

Effect of Dietary Potassium on Growth,Nitrogen Metabolism,Osmoregulation and Immunity of Pacific White Shrimp （Litopenaeus vannamel） Reared in Low Salinity Seawater

An 8 weeks feeding experiment was conducted to determine the effect of dietary potassium on the growth and physio-logical acclimation of Pacific white shrimp （Litopenaeus vannamei） reared in diluted seawater （salinity 4）. Six semi-purified practical diets containing 0.59, 0.96, 1.26, 1.48, 1.74, and 2.17 g potassium K＋ per 100 g diet were formulated, respectively. The survival and feed conversion rate did not show significant difference among groups of shrimps given these diets （P〉0.05）. The shrimps fed the diets containing 0.96-1.48 g K＋ per 100g diet gained the highest weight, specific growth rate, and protein efficiency ratio. Their ammonium-N excretion rate as well as hemolymph concentration of Na＋ and C1- were significantly lower than those of the control （P〈0.05）, but a reverse trend was observed for their gill Na＋/K＋-ATPase. Moreover, the shrimps fed with 1.48 g K＋ per 100 g diet were the highest in hemolymph urea level, and the phenoloxidase and lysozyme activities were significantly higher than those of the control （P〈0.05）. The growth and physiological response of the test shrimps suggested that diet containing 1.48 g K＋ per 100 g diet improved the growth of L. vannamei in low-salinity seawater, and enhanced the physiological acclimation of the organism.     

三丁基锡对罗氏沼虾血清中免疫酶活力的影响

将罗氏沼虾(Macrobrachium rosenbergii)在含0.1、0.2、0.4 mg/L三丁基锡(TBT)的水体中浸浴48 h,检测TBT对罗氏沼虾血清中部分免疫酶活力的影响。结果表明:12 h内高剂量(0.4 mg/L)可诱导超氧化物歧化酶(SOD),酶活力差异有统计学意义(P0.01),至48 h时,诱导作用逐步解除;12 h内各剂量组血清中酸性磷酸酶(ACP)活力均表现抑制效应,酶活力差异有统计学意义(P0.05),而高剂量组在48 h时TBT对ACP表现为诱导效应,酶活力高于对照组,差异有统计学意义(P0.05);低剂量组(0.1 mg/L)TBT对罗氏沼虾血清中AKP活力的影响无统计学意义(P0.05),而中剂量组(0.2 mg/L)和高剂量组(0.4 mg/L)AKP活力表现为先抑制后逐渐转为诱导的效应,。     

免疫遗传算法在土石坝筑坝粗粒料本构模型参数反演中的应用研究

建立了确定筑坝粗粒料本构模型参数的有限元计算理论与IGA相结合的反演分析方法,利用公伯峡筑坝现场大型载荷试验资料,对筑坝原级配料的本构模型参数进行了反演研究。结果表明,由于在微弱风化花岗岩掺加云母片岩,颗粒破碎严重,爆破堆石料（3BⅠ）的原级配粗粒料反演参数远低于室内三轴试验成果;与3BⅠ堆石料相反,砂砾料（3BⅡ）现场反演得到的邓肯E~B模型参数高于室内三轴试验值;室内三轴试验成果不能客观反映筑坝材料的力学特性,对于当今的300 m级高土石坝建设,宜加强对原级配粗粒料的力学特性研究;同时对原型实测的空间位移场不同拟合方式进行了探讨,认为仅考虑铅直向变形的IGA参数反演成果能基本满足土石坝工程的精度要求。     

溶藻弧菌免疫胶体金快速检测试纸条的制备

采用溶藻弧菌ATCC17749菌体作为抗原免疫新西兰大白兔,制备兔抗溶藻弧菌多克隆抗体;利用柠檬酸钠还原氯金酸法制备胶体金及胶体金-抗体结合物,并将其喷涂在玻璃纤维膜上冷冻干燥;在硝酸纤维素膜上包被兔抗溶藻弧菌多克隆抗体和羊抗兔IgG分别作为检测线和控制线;将处理后的玻璃纤维素膜、硝酸纤维膜、样品垫及吸水纸组装成双抗夹心试纸条,并对试纸条灵敏度、特异性、稳定性进行评价。结果表明,制备的兔抗溶藻弧菌多克隆抗体的效价可达1∶512 000;胶体金溶胶的最佳制备条件为:加入1.8 mL 10 mg/mL的柠檬酸三钠,500 W微波炉中火加热10 min;试纸条检测线和质控线的抗体最佳包被量分别为8.0 mg/mL和1.0 mg/mL;试纸条检测灵敏度为1×106cfu/mL,特异性试验显示,试纸条与哈氏弧菌、副溶血弧菌、霍乱弧菌、拟态弧菌、美人鱼弧菌、弗氏弧菌、河流弧菌、创伤弧菌等8株菌无交叉反应;稳定性试验表明,试纸在4℃干燥条件下能稳定保存5个月以上。制备的溶藻弧菌多克隆检测试纸条可灵敏、较特异地检测源自病鱼的溶藻弧菌,整个检测过程可在10 min内完成,适用于溶藻弧菌的快速检测。     

海豚链球菌3种免疫原对牙鲆血细胞吞噬及SOD、POD和ACP活力的影响

本文制备海豚链球菌(Streptococcus iniae)强毒株NUF849的福尔马林灭活全菌(FKC)、胞外产物(ECPs)以及全菌与胞外产物的混合物(FKC+ ECPs)3种免疫原,以其免疫牙鲆(Paralichthys olivaceus),并在免疫牙鲆后第42天分别以海豚链球菌NUF849和NUF812攻毒.在免疫牙鲆后第7、14、21、28、35、42天及攻毒后第7天分别取样,测定血细胞吞噬能力及血清中超氧化物歧化酶(SOD)、过氧化物酶(POD)和酸性磷酸酶(ACP)的活力,以比较3种免疫原对这些免疫指标的影响.结果显示,免疫后尤其攻毒后,红细胞、淋巴细胞、单核细胞、嗜中性粒细胞等的吞噬率与吞噬指数均上升,FKC+ ECPs组显著高于其他组(P＜0.05);各免疫组SOD活力随时间延长而下降并趋于稳定,攻毒后各组数值有不同程度的升高,但FKC+ ECPs组数值最低;免疫组POD活力先升高,第28天起下降,攻毒后各组均下降,FKC+ ECPs组始终高于其他免疫组(P＜0.05);免疫组ACP活力也在免疫后第28天达到峰值,以某株NUF849攻毒后FKC+ ECPs组水平上升(相对于免疫后第42天),其他组显著下降;FKC、ECPs、FKC+ ECPs对菌株NUF849的相对免疫保护宰分别为50％、40％和90％,对NUF812的相对免疫保护率分别为60％、30％和80％,FKC+ ECPs组显著高于其他组(P＜0.05).以上研究表明,各免疫组均诱导了牙鲆的非特异性免疫应答,具有一定的免疫保护效果,其中FKC+ECPs组效果最佳.