三十烷醇对铜藻生长及岩藻黄素含量的影响

为探讨三十烷醇对铜藻生长的影响,本研究采用不同浓度(0、0.1、 0.5、1.0 和2.0 mg/L)的三十烷醇连续施用20 d,或用三十烷醇浸泡处理24 h后,再恢复正常条件培养20 d,分别测定了铜藻的生长、生理生化指标(光合色素、可溶性蛋白、可溶性糖)以及岩藻黄素含量的变化情况。结果显示,连续施用5 d时,各组藻体的比生长速率(RGR)、叶绿素a(Chl.a)、类胡罗卜素(Car)、可溶性蛋白、可溶性糖的含量均显著高于对照组;15 d时,2.0 mg/L浓度组的铜藻生长受到抑制,各项生理指标均显著下降;20 d时,除了0.1 mg/L浓度组的铜藻生长仍有促进外,其他3个较高浓度的施用组均受到不同程度的抑制,且三十烷醇的浓度越高,抑制作用越明显。经三十烷醇浸泡处理的藻体恢复正常条件培养5 d时,各组藻体的RGR、Chl-a、Car、可溶性蛋白和可溶性糖的含量均显著上升;10 d时,0.5和1.0 mg/L浓度组的铜藻生长速率仍显著高于对照组,但15 d时已与对照组无显著差异,且综合各项生理生化指标,1.0 mg/L组的促进作用最为明显,这种促生长的作用能持续到处理后10 d左右。岩藻黄素的含量除连续施用2.0 mg/L组在实验20 d时低于对照组外,其余各实验组在培养20 d内均显著高于对照组,其中1.0 mg/L三十烷醇浸泡处理24 h恢复培养10 d,可使岩藻黄素的含量提高79.5%。研究表明,连续施用低浓度的三十烷醇(0.1 mg/L) 20 d或以1.0 mg/L三十烷醇每10 d浸泡处理24 h,对铜藻的生长和岩藻黄素的积累有显著的促进作用。     

岩藻黄素的生物活性及应用研究进展

岩藻黄素(fucoxanthin)是一类类胡萝卜素,主要分布在褐藻和硅藻中,并且结构已经被确定。岩藻黄素具有抗癌、抗炎、减肥等生物活性。利用这些功能,岩藻黄素在临床疾病防治以及生物工程等方面展示出十分广阔的应用前景。     

体外培养三角帆蚌外套膜细胞的生物学特性及有核珍珠培育

通过光学显微镜、透射电镜、流式细胞仪等方法对体外培养的三角帆蚌(Hyriopsis cumingiiLea)外套膜细胞进行观察和检测,证明其具有与蚌体内细胞相同的生物学结构、分裂增殖能力和分泌珍珠质的生物活性。在此基础上,对三角帆蚌有核珍珠培育技术进行探讨。将培养细胞黏附在表面经过促黏壁物质处理的珠核表面,再插入育珠蚌体内培育,120 d后,蚌体内可形成完备的珍珠囊,并在珠核表面有明显的珍珠质沉积;6个月后,珍珠质可将整个珠核包裹起来形成有核珍珠。本研究初步证明了细胞法培育有核珍珠的可行性。[中国水产科学,2007,14(1):149-154]     

穿膜肽引导的体外表达转录因子蛋白Sox2进入红鳍东方鲀精巢细胞系

为了探索适用于体外培养的鱼细胞外源基因转入方法, 本研究通过构建红鳍东方鲀(Takifugu rubripes)转录因子Sox2的重组表达载体pET32a(+)-Sox2-11R-6His, 诱导表达并纯化得到了C末端连接多聚精氨酸(11R)的重组蛋白Sox2-11R-6His, 以其与红鳍东方鲀精巢细胞系细胞共孵育12 h后, 光镜观察结合Western Blot检测发现重组蛋白进入细胞的效率与浓度呈剂量依赖关系且最佳孵育浓度为8 μg/mL, 当重组蛋白质量浓度达到10 μg/mL时, 表现出明显的细胞毒性。对外源蛋白进行免疫荧光标记定位, 发现重组蛋白分布于细胞质, 部分进入到细胞核中。证明了穿膜肽11R可以有效运载转录因子重组蛋白至红鳍东方鲀的细胞系细胞中。本研究旨在将广泛应用于哺乳动物的细胞基因递送载体穿膜肽应用于鱼类细胞系细胞。

     

天然叶黄素对黄颡鱼生长性能和皮肤着色的影响

研究了天然叶黄素(源自万寿菊,含量为4.64％)的添加水平对黄颡鱼生长和皮肤着色效果的影响.在黄颡鱼基础饲料中,分别添加0％、0.15％、0.30％、0.60％、8.00％的受试物,在不影响饲料营养水平的基础上设计最高剂量组为可能摄入剂量组的10倍以上,经挤压熟化工艺加工后,实际检测叶黄素含量为未检出(检出限=0.5 mg/kg)、24.2、54.2、118.0、1 700.0mg/kg,依次命名为C、L24.2、L54.2、L118、L1700.饲养90 d后发现,添加天然叶黄素的4个剂量组的特定生长率和饲料效率、L24.2组的蛋白沉积率、L54.2组和L118组及L1700组的能量沉积率均显著高于对照组(P＜0.05).体侧和腹部皮肤的黄度(b*)和饱和度(C*)随饲料中天然叶黄素含量的增加而逐步升高,L118组可达到饱和,并与L1700组之间没有显著差异(P＞0.05),L118组和最高剂量组皮肤中黄色素过度沉积,并向肌肉中转移.皮肤中叶黄素的蓄积系数呈现先升高后下降的趋势,最高为76.25 mg/kg饲料.结果表明,24.2～1700mg/kg的天然叶黄素显著提高了初始体质量21 9的黄颡鱼的生长性能.天然叶黄素作为黄颡鱼皮肤着色剂的最适剂量为76.25 mg/kg饲料.     

The incorporation and metabolism of polyunsaturated fatty acids in phospholipids of cultured cells from chum salmon (Oncorhynchus keta)

The incorporation and metabolism of (n-3) and (n-6) polyunsaturated fatty acids were studied in a cell line derived from chum salmon heart (CHH-1). Supplementing media with 25 M fatty acid considerably altered the cellular fatty acid composition but did not affect the lipid class composition or cause the appearance of cytoplasmic lipid droplets. CHH-1 cells exhibited considerable -6-desaturase activity but showed no preference between (n-3) and (n-6)PUFA substrates. CHH-1 cells also possess -5-desaturase activity which showed preference towards (n-3)PUFA, but -4-desaturase activity was totally absent. Elongation of 20-carbon PUFA was especially active in CHH-1 cells with 22-carbon PUFA being specifically incorporated into PE and PS lipid classes. The fatty acid composition of PI indicated specific incorporation of 20-carbon PUFA into this lipid class. Supplementation with 22:6(n-3) generated fatty acid compositions more closely resembling those of intact salmonid hearts. Substantial chain shortening of 22:6(n-3) to 20:5(n-3) occurred.Abbreviations BHT butylated hydroxytoluene - BSA bovine serum albumin - CL cardiolipin - FCS fetal calf serum - PA phosphatidic acid - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - PUFA polyunsaturated fatty acid - SM sphingomyelin     

Cytotoxicity of Streptococcus agalactiae secretory protein on tilapia cultured cells

Streptococcus agalactiae secrete virulence factors believed to be able of killing host tissues, especially under elevated water temperature. A direct effect of S. agalactiae secretory products on tilapia cells was tested on the tilapia kidney (TK-1) cell culture. The bacteria were cultured under four different temperature levels: 22, 29, 32 and 37°C; the cell-free portion was processed through SDS-PAGE; and distinct bands were identified by LC-MS/MS. At least, three virulence factors were identified, Bsp, PcsB and CAMP factor, with increasing levels as the cultured temperature rose. Expressions of bsp, pcsB and cfb were also up-regulated with the rising of the temperature in S. agalactiae culture. The supernatant from the bacteria cultured under specified temperatures was added into TK-1 cell-cultured wells. Morphological damage and mortality of the cultured cells, as determined by MTT method, were increased progressively from the supernatant treatment according to the rise of temperature in S. agalactiae culture. This study suggests that the production of the three virulence factors of S. agalactiae reported herein is temperature-dependent, and it is likely that CAMP factor directly kills the TK-1 cells since the other two types of protein are involved in S. agalactiae cell division and the bacterial adherence to host tissues.     

鲟鱼硫酸软骨素对小鼠免疫器官肥大细胞的影响

为研究鲟鱼硫酸软骨素对小鼠免疫器官的影响,连续对小鼠灌胃不同剂量鲟鱼硫酸软骨素2周后,外科手术取出胸腺和脾脏,并依次进行称重、卡洛氏固定、石蜡包埋、组织切片、甲苯胺蓝染色,40倍物镜下观察鲟鱼硫酸软骨素对小鼠免疫器官肥大细胞数量的影响。结果表明：鲟鱼硫酸软骨素能促进小鼠免疫器官重量指数增大,并能显著促进免疫器官中肥大细胞数量的增长。其中,600 mg/kg剂量下小鼠胸腺指数显著( P<0．05)增大,胸腺中肥大细胞数量极显著( P<0．01)增加,60 mg/kg剂量下小鼠脾脏重量的增加最为明显( P<0．01),6 mg/kg剂量下小鼠脾脏中肥大细胞数量的增加最为明显( P<0．01)。     

Polyunsaturated fatty acid metabolism in cultured fish cells: Incorporation and metabolism of (n-3) and (n-6) series acids by Atlantic salmon (Salmo salar) cells

The incorporation and metabolism of (n-3) and (n-6) polyunsaturated fatty acids (PUFA) supplemented to growing cultures were studied in Atlantic salmon (AS) cells. A fatty acid concentration of 25 μM considerably altered the fatty acid composition of AS cells without increasing the neutral lipid content of the cells or inducing the production of cytoplasmic lipid droplets. Whereas Δ6 and Δ5 desaturase activities were significantly expressed in AS cells, Δ4 desaturase activity was very low. Both the Δ6 desaturase activity and the Δ5 desaturase activity showed some preference for (n-3) PUFA.     

Application of Two-Level Full Factorial Design for the Extraction of Fucoxanthin and Antioxidant Activities from Sargassum siliquosum and Sargassum polycystum

Two-level full factorial design was employed to identify the extraction parameters that can improve the derivation of fucoxanthin content (FC), total carotenoid content (TCC), and antioxidant from two brown seaweeds, Sargassum siliquosum (SS) and Sargassum polycystum (SP). These parameters included temperature (A: 4–45°C), time (B: 30–1,440 min), and solvent-to-solid ratio (C: 10–50 ml/g). Antioxidant activities were determined as trolox equivalent antioxidant capacity (TEAC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity, and ferric-reducing antioxidant power (FRAP). Results showed that all three factors were significant (p < 0.05) in providing higher FC in both species. These factors were also significant in obtaining higher TCC in SS; whereas in SP, TCC was only affected by solvent-to-solid ratio. Only temperature was found to contribute significantly to a higher TEAC in both species. However, none of the factors improved DPPH for SS, except temperature and time for SP. For SS, only time was significant in obtaining higher FRAP; whereas temperature and time were significant for SP. Hence, results indicate that a simple modification in the extraction temperature, time, and solvent-to-solid ratio will be able to improve the derivation of fucoxanthin, carotenoids, and antioxidant activities.     

Incorporation and metabolism of (n-3) and (n-6) polyunsaturated fatty acids in phospholipid classes in cultured turbot (Scophthalmus maximus) cells

The incorporation and metabolism of various (n-3) and (n-6) polyunsaturated fatty acids (PUFA) supplemented to the culture medium was investigated in a turbot cell line (TF). The distribution, and the occurrence and extent of further metabolism of incorporated PUFA via desaturation/elongation mechanisms in specific phospholipid classes was determined from the different fatty acid compositions. The cells contained Δ6 and Δ4 desaturase activities but were generally deficient in C18–20 elongase activity. Δ5 Desaturase activity was generally masked by this deficiency but was present. The compositional data indicated that there was a high degree of specificity between individual phospholipid classes and particular fatty acids probably driven by the specificities of the acylating enzymes. The highest percentages of the supplemented acids were generally observed in the phosphatidic acid/cardiolipin fraction (PA/CL), suggesting a role for PA in the incorporation of the supplemented acids into the phospholipid pool. PI had a characteristic composition consistent with a putative role as a pool of precursor fatty acid for eicosanoid synthesis. Mechanisms were evident for generating and/or maintaining this composition.     

不同因子对花鲈胚胎干细胞增殖的影响

胚胎干细胞(ES细胞)是从动物早期发育胚胎中分离出来的具有发育全能性的一种未分化细胞系。维持花鲈胚胎干细胞(LJES1)的体外生长、增殖及未分化状态需要在培养基中添加一些生长因子。实验通过配制省略1种或多种因子的培养基PESM0～10，计数LJES1细胞在各培养基中增殖的数量，确定各种生长因子对LJES1细胞的增殖作用。同时，对LIF因子和bFGF因子的作用进行了重点的研究，发现LIF因子对早期的LJES1细胞增殖几乎没有作用，其主要作用是维持LJES1细胞的未分化状态，但对晚期LJES1细胞的增殖有一定的作用；bFGF因子对LJES1细胞有强烈的刺激增殖的作用；鲈鱼胚胎抽提液(PEE)以及鲈鱼血清(FS)也促进了LJES1细胞的增殖，2-巯基乙醇(2-ME)具有还原血清中的含硫化合物、防止过氧化物对LJES1细胞的损害及促进贴壁的作用，因而也促进了LJES1细胞的增殖。     

Incorporation and metabolism of (n-3) and (n-6) polyunsaturated fatty acids in phospholipid classes in cultured rainbow trout (Salmo gairdneri) cells

The incorporation and metabolism of various (n-3) and (n-6) polyunsaturated fatty acids supplemented to the culture medium was investigated in the rainbow trout cell line, RTG-2. The distribution, and the occurrence and relative extent of further desaturation and elongation of the incorporated acids was determined in individual phospholipid classes by analysis of the fatty acid compositions. RTG-2 cells exhibited Δ6 and Δ5 desaturase activities whereas Δ4 desaturase activity was almost totally absent. The percentage of precursor acids was greatest in the phosphatidic acid/cardiolipin fraction (PA/CL), suggesting a role for possibly PA in the initial incorporation of these acids into the phospholipid pool. The compositional data indicated that individual intermediates and products of the desaturation pathways were associated with specific phospholipid classes probably via mechanisms depending upon the specificities of the acylating enzymes. The composition of phosphatidylinositol (PI) and the tightly controlled mechanisms for generating/maintaining it are consistent with a role for this phospholipid in providing precursor fatty acid for eicosanoid synthesis.     

供体细胞的不同处理及重组胚化学激活对体细胞克隆胚胎发育的影响综述

哺乳动物体细胞克隆经过几年的发展,已取得了很大进展。已有多种细胞被采用并得到了克隆后代。但动物克隆效率仍然很低,供体细胞的选择和重组胚激活是动物克隆中的重要步骤。供体细胞的细胞周期、细胞类型、冷冻处理和化学激活等方面都可能影响动物克隆的效率。     

GnRH_A和紫萁抑制大黄鱼性腺早熟的机制

应用脑垂体组织生理学和免疫组织化学方法，对GnRH-A和紫萁抑制大黄鱼性早熟的作用机制进行了研究。结果表明，性早熟大黄鱼脑垂体许多促性腺激素分泌细胞的胞质出现空泡，提示性早熟的原因可能是由于GtH细胞提早进入分泌活动所致。长期服用促性腺激素释放激素类似物的养殖大黄鱼（没有性早熟），它的脑垂体GtH细胞对GnRH抗独特型发生弱的免疫阳性反应，而对照组性早熟鱼仍出现强的反应，表明实验组大黄鱼脑垂体GtH细胞对GnRH-A的应答能力下降，出现脱敏效应，这可能与GnRH-A抑制大黄鱼性早熟有关。养殖大黄鱼长期服用紫萁，它的脑垂体GtH细胞膜上GnRH受体则没有出现脱敏现象，提示紫萁抑制大黄鱼性早熟的机制不同于GnRH类似物，确切机制有待进一步研究。     

脊椎动物源细胞因子对合浦珠母贝外套膜细胞培养及其矿化功能的影响

筛选出能促进合浦珠母贝外套膜细胞培养及矿化功能的细胞因子,以作为优化本物种细胞培养基的参考,挑选三龄合浦珠母贝,获取其外套膜进行原代细胞培养。向各实验组细胞培养基中分别添加表皮生长因子(EGF)、内皮细胞生长添加剂(ECGS)、胰岛素样生长因子-1(IGF-1)和碱性成纤维细胞生长因子(bFGF),通过比较细胞活性、贴壁能力、迁移能力和4种基质蛋白基因(pif80、n16、msi7及accbp)表达水平的变化,来评判这些因子对细胞培养及细胞矿化功能的影响。结果显示:(1)EGF能显著提高原代培养细胞的活性、贴壁能力和迁移能力,并促进pif80、n16和accbp基因的表达;(2)ECGS能增强细胞的贴壁能力和迁移能力,并大幅提高pif80、n16和msi7基因的表达水平;(3)IGF-1能显著增强细胞活性、贴壁能力、迁移能力和msi7的基因表达水平,但对pif80、n16和accbp基因的表达有一定抑制作用;(4)碱性成纤维细胞生长因子能增强细胞的活性及贴壁能力,并显著提升pif80、n16和accbp基因的表达水平。研究表明,脊椎动物源细胞因子具有延长细胞培养时间、增强细胞活性及促进矿化相关基因表达的作用,能够用于优化合浦珠母贝外套膜细胞的培养基。     

人工养殖不同年龄史氏鲟的血液学参数

测定了5个年龄组人工养殖史氏鲟的血液学参数,其中红细胞（RBC）、红细胞比积（HCT）、血红蛋白（HGB）、平均红细胞血红蛋白含量（MCH）、平均红细胞血红蛋白浓度（MCHC）、白细胞（WBC）等随年龄的增长而上升,WBC、HCT5年龄组与1～4龄各年龄组间有显著和极显著差异（P〈0．05）;HGB、MCH、MCHC相邻组间无显著性差异;平均血红细胞体积（MCV）、血栓细胞（PLT）、血红蛋白含量分布宽度（HDW）等1～4龄变化不大,仅在5龄MCV、PLT有较显著的上升,而HDW有显著的下降;红细胞体积分布宽度（RDW）则保持相对稳定,各年龄组间无显著差异;平均血栓细胞体积（MPV）变化不显著,随年龄增长呈下降趋势。在白细胞分类计数DLC值变化中,白细胞中各类细胞比例依次为淋巴细胞（Lym）〉嗜碱性粒细胞（Baso）〉嗜中性粒细胞（Neut）〉单核细胞（Mon）,随着年龄的增长,Lym的含量逐渐下降,而Baso、Neut和Mon均呈上升趋势。     

The effect of culture on morphology, lipid and fatty acid composition, and polyunsaturated fatty acid metabolism of rainbow trout (Oncorhynchus mykiss) skin cells

Rainbow trout (Oncorhynchus mykiss) skin cell cultures were obtained by trypsinization of the tissue and grown in Leibovitz L-15 medium. Lipid class compositions, and fatty acid profiles of total lipids and individual phospholipid classes were determined at different times of culture. The metabolism of polyunsaturated fatty acids (PUFA) was investigated by incubating primary cultures after 7 and 14 days with [1-¹⁴C]18:2n-6 and [1-¹⁴C-]18:3n-3. The change in morphology between epithelial-like primary cultures and fibroblastic-like secondary subcultures was accompanied by alterations in the lipid composition. Polar lipids became predominant by 14 days in culture. The relative proportions of phosphatidylcholine (PC), the most abundant phospholipid, phosphatidylinositol and cholesterol increased significantly, while sphingomyelin decreased. Saturated fatty acids, 18:1n-9, n-6 and n-9PUFA were more abundant in total lipid in cultures at 14 days and 4 months than in cells initially isolated which contained higher percentages of longer chain monoenes and n-3PUFA. The changes in fatty acid composition with time in culture were observed in all the major phospholipid classes. Rainbow trout skin cells in culture desaturated and elongated both 18:2n-6 and 18:3n-3, with 20:4n-6 and 20:5n-3 being the most abundant products, respectively. PC presented the highest incorporation of radioactivity, especially following incubation with 18:3n-3. Lipid metabolism in general increased with the age of primary cultures, with both the amount of C18 PUFA incorporated and metabolized by desaturation/elongation significantly increased in 14 day cultures compared to 7 day cultures. Product/precursor ratios calculated for both n-6 and n-3 fatty acids showed that, while 6 desaturase activity was increased significantly with cell age, 5 desaturase activity was more affected by the fatty acid series, with 18:3n-3 being more readily transformed to 20:5n-3 than 18:2n-6 to 20:4n-6. Further desaturation of 20:5n-3 to hexaenes was low. Overall, the data suggested that the trout skin cell cultures were more similar to mammalian skin fibroblasts than mammalian epidermal/keratinocyte cultures.     

The effect of different carotenoid sources on skin coloration of cultured red porgy (Pagrus pagrus)

This study presents data on the effect of carotenoid sources on skin coloration of red porgy (Pagrus pagrus). Three experiments were conducted: in the first, fish were fed an astaxanthin (Naturose^®)‐supplemented diet, while the second fish received diets supplemented with β‐carotene (Rovimix β‐caroten^®) or lycopene (Lyc‐O‐Mato^®): Carotenoids were added to the level of 100 ppm in each diet, while a non‐carotenoid‐supplemented diet served as a control. In the third experiment, the effect of dietary protein/carbohydrate ratio on melanin content in the skin was investigated. For this experimentation, four diets were formulated to contain 50/23, 40/32, 30/48 and 20/59 protein/carbohydrate ratio. Naturose^® astaxanthin increased total carotenoid content in the dorsal skin area while β‐carotene and lycopene seem to have had no significant effect. Naturose^® was the only carotenoid source that had a significant effect on skin hue, promoting a reddish coloration to the dorsal skin area and a ventral hue similar to wild red porgy. No apparent effect of carotenoid source on skin melanin content was observed. In contrast, dietary protein/carbohydrate ratio affected melanin content in the skin. The fish fed the 50/23 diet showed significantly higher values. Farmed red porgy had eight times higher dorsal‐skin melanin content than wild ones.