油酸诱导建鲤(Cyprinus carpio var. Jian)肝细胞脂肪变性模型的建立

通过对油酸诱导时间及有效作用浓度的研究,建立油酸致建鲤肝细胞脂肪变性模型。本研究以胰蛋白酶消化法获得的原代肝细胞作为实验材料,加入不同浓度油酸(0、0.05、0.1、0.2、0.4、0.8 mmol/L)与肝细胞共培养24–48 h,诱导肝细胞脂肪变性,分别收集不同时段的肝细胞及上清液,采用试剂盒测定肝细胞中甘油三酯(TG)、总胆固醇(TC)的含量,同时测定上清培养液中谷丙转氨酶(GPT)、谷草转氨酶(GOT)、γ-谷氨酰转肽酶(γ-GT)、乳酸脱氢酶(LDH)和超氧化物歧化酶(SOD)的活性,MTT法测定肝细胞的存活情况,确定油酸最佳诱导浓度,油红O染色法观察细胞内脂肪滴的形成情况。研究结果显示,0.4 mmol/L油酸与肝细胞共培养48 h,可以显著提高肝细胞内TG、TC含量(P﹤0.05或P﹤0.01),极显著提高上清培养液中GOT、GPT、LDH、γ-GT的活性,显著降低上清培养液中SOD的活性,但与24 h相比有升高趋势,0.4 mmol/L油酸与肝细胞共培养24 h,光镜下可见细胞内有脂肪滴形成,以48 h最明显。综上所述,油酸浓度0.4 mmol/L、诱导时间48 h成功构建了肝细胞脂肪变性模型,为研究鱼类脂肪肝类疾病奠定了基础。     

鲤科骄子——建鲤(Cyprinus Carpio Var.jian)

<正> 建鲤是采用荷元鲤F_4长型品系与两个同型的雌核发育系相结合,经选择横交固定,六代定向选育,首次育成,遗传性状稳定(达99％以上)并能自繁自育的鲤鱼优良品种。建鲤的乳酸脱氢酶、同工酶、酯酶同工酶和血清蛋白电泳分析结果表明:它具有独特的生化遗传特征。建鲤生长优势非常显著。其体重增长较荷包红鲤、沅江鲤和荷元鲤分别高58.2～71.3％、44.5～49.1％和28.5～29.3％;群体增重分别高60.1～71.8％、49.1～50％和29.4～30.8％,并显著地高于其它鲤鱼和杂交鲤,能增产30％以上。     

裂壶藻对津新鲤(Cyprinus carpio var. Jian)生长、血液非特异性免疫指标及抗病力的影响

选用初始体重为(26.77±1.56)g的津新鲤(Cyprinus carpio var.Jian) 540尾,随机分为6组,每组3个重复,分别投喂含0、0.40％、0.80％、1.20％、1.60％和2.00％裂壶藻的等氮等能饲料,饲养56 d,探讨饲料中不同水平的裂壶藻对津新鲤生长、血液非特异性免疫指标及抗病力的影响.结果显示,在0.80％裂壶藻水平组中,增重率和特定生长率均显著提高(P＜0.05),肥满度则在1.20％水平组中达到最大值(P＜0.05),各组间成活率差异不显著(P＞0.05).不同水平的裂壶藻均可以显著提高津新鲤的抗氧化能力,其中过氧化氢酶、总超氧化物歧化酶和谷胱甘肽的含量均呈先上升后下降的趋势,且分别在1.20％、0.80％和1.20％组中达到最高(P＜0.05);而丙二醛含量呈先下降后上升的趋势,在0.80％组中达最低(P＜0.05).不同水平的裂壶藻均可以显著提高津新鲤的非特异性免疫力,溶菌酶呈先上升后下降的趋势,其中1.20％组活性最高(P＜0.05).一氧化氮含量随裂壶藻水平增加而增加,各添加组含量均显著高于对照组(P＜0.05).谷草转氨酶/谷丙转氨酶在0.80％组中达到最小值(P＜0.05).随着裂壶藻水平的增加,津新鲤血细胞呼吸爆发活性和免疫保护率得到显著提高,感染嗜水气单胞菌后的累计死亡率则显著降低(P＜0.05),其中,以0.80％组最佳.综合生长、血液非特异性免疫指标和抗病力来分析,饲料中裂壶藻的适宜添加水平为0.80％-1.20％.     

天津地区养殖白鲢Hypophthalmichthys molitrix (Cuvier et Valenciennes)、建鲤(Cyprinus carpio var.jian)染色体核型(karyotype)调查与分析

采用活体注射PHA肾细胞培养法和外周血淋巴细胞培养法进行染色体制片,对天津地区养殖鲢、建鲤进行核型调查与分析,评价染色体数目及核型指标在养殖鱼类种质鉴定中的作用。调查结果表明,分布在不同养殖区的种质状况各不相同的12个采样点的养殖鲢、鲤样本染色体数目恒定,分别为2n=48和2n=100;各个采样点的养殖鲢、建鲤样本与以往相比在核型上均没有发生明显改变,这说明核型是种的标志特征,但与种质状况无明显的相关性。     

喹乙醇诱导鲤肝细胞凋亡的研究

细胞凋亡(apoptosis)是受基因控制的细胞自主的有序死亡。凋亡细胞的形态特征主要表现为细胞核的染色质浓缩,边移,聚集在核膜下,进而核发生裂解,核碎片与细胞碎片有单层膜包裹形成凋亡小体(apoptosisbody)[1]。目前已发现某些物理因素(辐射、高温等)、细胞因子、病毒感染和     

CCl4对建鲤肝细胞DNA的毒性作用及对CYP3A基因表达的影响

四氯化碳(CCl₄)作为一种经典的肝脏毒物,被广泛应用于哺乳动物的肝损伤模型构建及保肝药物筛选。低、中、高浓度的CCl₄橄榄油溶液(6.25%、12.50%和15.00%,0.01 mL/g)腹腔注射建鲤72 h后,采用单细胞凝胶电泳和肝组织病理切片技术,测定血清中肝损伤生化酶来考察CCl₄对肝细胞DNA的毒性作用;同时采用实时荧光定量PCR法测定肝组织中CYP3A表达量的变化。结果显示,6.25%CCl₄作用建鲤72 h后肝组织切片检查没有发生明显的改变,对血清中酶学指标无显著影响,CYP3A的mRNA表达量与空白对照组相比也没有显著变化,而彗星实验结果显示该浓度的CCl₄作用肝细胞后,尾长、TDNA、尾矩及Olive 尾矩等DNA损伤指标与对照组相比,显著增大;随着CCl₄作用浓度的增加,肝细胞肿胀、广泛空泡变性,出现核固缩和核溶解等组织学的变化,12.50%和15.00% CCl₄均能显著引起血清中谷丙转氨酶(GPT)和谷草转氨酶(GOT)水平升高,15.00% CCl₄能显著促进乳酸脱氢酶(LDH)和丙二醛(MDA)大量生成;彗星实验中各项损伤指标也随着染毒剂量增加而极显著增大;同时,12.50%和15.00% CCl₄组的CYP3A mRNA表达量显著降低。结果表明,CCl₄对建鲤肝细胞DNA具有毒性作用,在实验的浓度范围内,CCl₄ 能抑制CYP3A mRNA表达;随着CCl₄作用浓度的增大,血清酶、病理切片和彗星实验结果表现出剂量效应,并有一定的一致性,彗星实验表现出更高的灵敏性。     

草鱼肝细胞脂变模型的建立及脂代谢基因表达分析

为了筛选草鱼肝细胞脂肪变性的最佳诱导剂及浓度,并初步分析脂肪乳剂(lipid emulsions,LE)引起草鱼肝细胞脂肪变性的作用机理,以草鱼(Ctenopharyngodon idellus)正常肝细胞为研究对象,建立草鱼脂肪变性肝细胞模型,以含10%胎牛血清的基础培养液为对照组,处理组为含20%脂肪乳剂0.5~2 m L/L和含20%、50%胎牛血清的诱导培养液,孵育草鱼肝细胞48 h后,定量分析肝细胞内的甘油三酯(TG)含量,观察脂滴积聚情况及肝细胞超微结构的变化,检测细胞培养上清中谷丙转氨酶(alanine transaminase,ALT)、谷草转氨酶(aspartate transaminase,AST)的活性,q RT-PCR技术检测脂代谢关键基因(PPAR?、PPAR?、SREBP-1c、LPL、Lep和UCP2)的转录水平变化,蛋白质印迹技术检测PPAR?、SREBP-1c的蛋白水平变化。结果发现,含1~2 m L/L LE的诱导液组和含20%、50%FBS的诱导液组与对照组相比TG含量均显著上升(P0.05),且20%FBS和各浓度LE诱导组的转氨酶活性与对照组相比差异不显著(P0.05),表明含1~2 m L/L LE的诱导液和含20%FBS的诱导液均可建立草鱼营养性脂肪肝细胞模型。在肝细胞脂变模型中,PPARγ和LPL等脂代谢基因的表达量显著升高(P0.05),而Lep基因表达量显著降低(P0.05),PPARγ和SREBP-1c的蛋白水平升高。结论认为:采用1~2 m L/L LE和20%的FBS均可以在短时间内建立草鱼肝细胞脂肪变性模型,含1 m L/L LE的诱导液诱导效果最佳;肝细胞内脂质的蓄积可能与脂肪代谢关键基因PPARγ、SREBP-1c、LPL及Lep等密切相关。     

水飞蓟素对建鲤肝细胞DNA损伤的保护作用及相关细胞因子的影响

为了评价水飞蓟素对建鲤肝组织损伤的保护作用,探索其可能的保肝机制,本研究用含水飞蓟素(0.1、0.5和1.0 g/kg)的饲料饲喂建鲤60 d,再腹腔注射30%四氯化碳(CCl4)植物油混合液,损伤72 h后采集肝组织,分别研究肝指数、肝细胞DNA损伤、肝组织病理切片、核转录因子NF-κB/C-Rel及细胞因子i NOS和IL-1β的mRNA表达量的变化。结果显示,0.5和1.0 g/kg的水飞蓟素能显著抑制CCl4导致的建鲤肝指数增大,同时能有效减少肝细胞DNA断片的产生,对肝细胞彗星的慧尾长、尾部DNA百分含量、尾矩及Olive尾矩等损伤指标均有显著改善作用;水飞蓟素能明显减轻CCl4作用导致的肝组织广泛性空泡变性、细胞轮廓不清、核固缩和核溶解等组织学病变;0.5和1.0 g/kg的水飞蓟素能显著抑制CCl4诱导的NF-κB/C-Rel和i NOS表达量的增加,而低、中、高浓度的药物均能显著下调IL-1β的表达量;随着水飞蓟素浓度的增大,对肝指数、肝细胞DNA损伤、病理切片及细胞因子的mRNA表达的影响均表现出剂量效应。结果表明,水飞蓟素能有效改善肝细胞DNA的损伤程度,其保肝作用可能与抑制NF-κB活化及下调其下游细胞因子i NOS和IL-1β的表达有关。     

有效微生物制剂(EM)对建鲤肠道菌群及其蛋白酶和肝胰脏蛋白酶活性的影响

本实验以有效微生物制剂(Effective Microorganisms, 简称EM)作为饲料添加剂饲养建鲤。45d后对建鲤肠道菌群和蛋白酶活性进行研究,结果发现:EM对饲养后的建鲤肠道菌群有明显的改善,实验组乳酸菌比对照组分别增加了125.10％、345.19％和488.50％。大肠杆菌比对照组分别减少了82.01％、92.45％和90.57％,定植效果良好;实验组的肠道蛋白酶活性比对照组高,分别提高6.18％、9.38％和17.70％。添加2％、4％EM的实验组的肝胰脏蛋白酶活性与对照组相近,而添加6％的实验组其肝胰脏蛋白酶活性比对照组提高5.78％,实验组建鲤生长加快,饵料系数比对照组分别降低了1.35％、7.74％和22.90％。     

鲈鲤(Percocypris pingi)染色体核型及C-带分析

鲈鲤(Percocypris pingi)为长江上游特有鱼类及四川省重点保护鱼类,为验证其倍性及丰富其细胞遗传学内容,采用植物性血凝素(PHA)体内注射肾细胞直接制片法研究鲈鲤的核型,采用改进的BSG法研究其C-带.结果显示,鲈鲤的染色体数目为98条,其核型公式为4n=98=42m+30sm+10st+16t,臂数NF=170.本研究发现,鲈鲤有16条端部着丝粒染色体,未发现小染色体及其他异型性染色体.鲈鲤C-带带纹较丰富,显示着丝粒带、端粒带和染色体居间带3种类型的C-带,部分染色体同时含有多种类型的C-带着色,如m2、st4同时含有以上3种C-带带纹类型.鲈鲤染色体异染色质含量高达(58.824±0.034)％.本研究再次确认鲈鲤为四倍体,同时指出其染色体具有一定的“可塑性”,反应出鲈鲤对于地区特化的生境具有较强的适应性.     

Dietary lysine requirement of juvenile Jian carp (cyprinus carpio var. Jian)

This study was designed to determine the dietary lysine requirement of juvenile Jian carp (cyprinus carpio var. Jian). A total of 990 juvenile Jian carps (initial weight 7.89 ± 0.04 g) were randomly allocated six groups of each three replicates, feeding isonitrogenous (320 g kg^?1 crude protein) and isolipidic (50 g kg^?1 crude fat) diets containing graded levels of coated l ‐lysine, with dietary lysine content ranging from 22 to 69 g kg^?1 of protein, respectively, for 80 days. Final weight, protein deposition and protein production value were significantly improved in response to the increasing levels of dietary lysine up to 59 g kg^?1 of protein and remained nearly constant thereafter. Whole‐body protein content followed a similar pattern as growth parameters in relation to dietary lysine level. Hepatopancreas and intestine protein content, hepatopancreas index (HSI) and intestine index (ISI) were positively affected by dietary lysine level. The dietary lysine requirement of juvenile Jian carp estimated by broken line analysis based on weight gain was 59 g kg^?1 of protein.     

Effects of dietary thiamin supplement on growth,body composition and intestinal enzyme activities of juvenile Jian carp (Cyprinus carpio var. Jian)

A total of 1050 juvenile Jian carp (Cyprinus carpio var. Jian) (8.20 ± 0.02 g) were fed diets containing seven graded levels of thiamin (0.25, 0.48, 0.79, 1.06, 1.37, 1.63 and 2.65 mg kg^?1) for 60 days to investigate the effects of thiamin on growth, body composition and digestive enzyme activities. Percent weight gain (PWG), feed intake and feed efficiency (FE) were the lowest in fish fed the basal diet (P < 0.05). Protein productive value and lipid productive value increased with increasing dietary thiamin levels up to 1.06 and 0.79 mg kg^?1 diet, respectively (P < 0.05). Body protein and lipid increased with increasing dietary thiamin levels (P < 0.05), while moisture and ash of fish carcasses decreased with the increase in dietary thiamin supplementation (P < 0.05). Intestinal folds height had a similar trend to PWG (P < 0.05). Activities of α‐amylase, lipase, trypsin, Na⁺, K⁺‐ATPase, alkaline phosphatase and gamma‐glutamyl transpeptidase in intestine were all affected by the dietary thiamin (P < 0.05). In conclusion, thiamin could improve growth and intestinal enzyme activities of juvenile Jian carp. The dietary thiamin requirement of juvenile Jian carp (8.0–60.2 g) based on PWG was 1.02 mg kg^?1 diet.     

Effect of dietary pantothenic acid supplement on growth, body composition and intestinal enzyme activities of juvenile Jian carp (Cyprinus carpio var. Jian)

A 9-week feeding trial was carried out with juvenile Jian carp to study the effect of dietary pantothenic acid (PA) on growth, body composition and intestinal enzyme activities. Semi-purified diets with seven levels (4.0, 15.5, 25.6, 36.1, 45.9, 56.1 and 65.9 mg PA kg⁻¹) of supplemental calcium d -pantothenate were fed to Jian carp (13.0 ± 0.0 g). PA improved specific growth rate (SGR), protein productive value (PPV), protein efficiency ratio (PER) and lipid production value (LPV) ( P <  0.05). Fish fed the control diet had significantly lower feed efficiency (FE) than that in any other group ( P <  0.05). Body protein content increased with increasing PA levels ( P <  0.05), but moisture, lipid and ash of fish carcasses were negatively related to the graded PA levels ( P <  0.05). Intestine protein content (IPC), hepatopancreas protein content (HPC) and activity of α-amylase, lipase, trypsin, Na⁺,K⁺-ATPase, alkaline phosphatase (AKP) and gamma-glutamyl transpeptidase (γ-GT) were all positively affected by the dietary PA levels ( P <  0.05), while intestine index (ISI) and hepatopancreas index (HSI) decreased with the increment of supplemental levels of PA ( P <  0.05). These results suggested that PA could enhance fish growth and intestinal enzyme activities. The dietary PA requirement of juvenile Jian carp, Cyprinus carpio var . Jian (13.0–73.0 g), for optimal growth estimated by the broken-line analysis was 23.0 mg PA kg⁻¹ diet.     

Immune response,disease resistance and intestinal microflora of juvenile Jian carp (Cyprinus carpio var. Jian) fed graded levels of pantothenic acid

This study was to investigate the effect of dietary pantothenic acid (PA) on the disease resistance, immune response and intestinal microflora on juvenile Jian carp (Cyprinus carpio var. Jian). Seven diets (4.0, 15.5, 25.6, 36.1, 45.9, 56.1 and 65.9 mg PA kg^?1) were fed to Jian carp (12.95 ± 0.03 g) for 9 weeks. After 9‐week feeding trial, the challenge experiment with Aeromonas hydrophila was conducted to determine the impact of PA on fish disease resistance. Survival rate after challenge was promoted with the increasing PA levels (P < 0.05). Blood counts also significantly increased up to the dietary PA level of 25.6 mg PA kg^?1 (P < 0.05). Leucocyte phagocytic activity, lectin potency, lysozyme and acid phosphatase activity, and total iron‐binding capacity were improved with increasing PA levels (P < 0.05). Serum immunoglobulin M level and agglutination antibody titre to A. hydrophila were increased (P < 0.05) in fish fed the diets with the dietary PA levels between 56.1 and 65.9 mg kg^?1. PA also promoted the growth and reproduction of Lactobacillus and depressed Escherichia coli and A. hydrophila (P < 0.05). These results suggested that pantothenic acid could improve disease resistance, immune response, and the balance of intestinal microflora in juvenile Jian carp.     

Effects of graded levels of dietary vitamin C on the growth,digestive capacity and intestinal microflora of juvenile Jian carp (Cyprinus carpio var. Jian)

A 60‐day experiment was carried out to study the effects of vitamin C [ascorbic acid (AA)] on the growth, digestive enzyme activities and intestinal microbial population. Diets with six levels (0.0, 21.4, 45.1, 69.5, 93.6 and 142.1 mg AA kg^?1 diet) of supplemental ascorbyl polyphosphate were fed to juvenile Jian carp (Cyprinus carpio var. Jian) (12.63±0.02 g). The specific growth rate (SGR), feed efficiency and productive protein value were significantly improved with increasing AA levels up to 45.1 mg AA kg^?1 diet (P<0.05). The hepatopancreas and muscle AA concentrations were increased with increasing dietary AA levels up to 69.5 and 45.1 mg kg^?1 diet respectively (P<0.05). The activities of intestinal trypsin, chymotrypsin, lipase, α‐amylase, Na⁺, K⁺‐ATPase, alkaline phosphatase, gamma‐glutamyl transpeptidase and creatinkinase were all positively affected by the AA supplementation (P<0.05). Intestinal Lactobacillus and Bacillus were increased with increasing AA levels up to 45.1 mg AA kg^?1 diet (P<0.05), while intestinal Escherichia coli decreased with increasing AA levels up to 45.1 mg AA kg^?1 diet (P<0.05). In conclusion, AA improve the digestive capacity and intestinal microbial population of Jian carp, and the dietary AA requirement for SGR of juvenile Jian carp was 40.9 mg AA kg^?1 diet.     

Dietary pyridoxine requirement of juvenile Jian carp (Cyprinus carpio var. Jian)

In a 80-day feeding trial, a total of 1050 juvenile Jian carp ( Cyprinus carpio var. Jian) with an average initial weight of 10.71 ± 0.05 g were fed semi-purified diets containing seven graded levels of pyridoxine (0.20, 1.71, 3.23, 4.96, 6.32, 8.58 and 12.39 mg pyridoxine kg⁻¹ diet). Results indicated that with increasing dietary pyridoxine levels up to 4.96 mg kg⁻¹ diet, percent weight gain (PWG) and specific growth rate (SGR) were improved, and no differences were found with further increase of pyridoxine levels. Feed intake also followed the similar pattern to that observed with PWG and SGR when dietary pyridoxine levels were ≤6.32 mg kg⁻¹ diet. But feed efficiency and protein efficiency ratio were not affected by pyridoxine levels. Crude protein of carcass, productive protein value and plasma ammonia concentration were improved with increasing dietary pyridoxine levels up to 4.96 mg kg⁻¹ diet. Amylase activities in the intestine were improved with increasing dietary pyridoxine levels up to 4.96 mg kg⁻¹ diet, but protease and lipase activities in the intestine were not affected by pyridoxine levels. Na⁺, K⁺-ATPase and Gamma-glutamyl transpeptidase activities in proximal intestine, mid intestine (MI) and distal intestine (DI) were lowest when fed the diet containing 1.71 mg pyridoxine kg⁻¹ diet. The alkaline phosphatase activities in MI and DI followed the same pattern. The dietary pyridoxine requirement of juvenile Jian carp based on PWG estimated by broken line model was 6.07 mg pyridoxine kg⁻¹ diet.     

Effect of dietary iron levels on growth,body composition and intestinal enzyme activities of juvenile Jian carp (Cyprinus carpio var. Jian)

A 60‐day feeding trial was carried out to investigate the effect of iron on growth, body composition and digestive enzyme activities. Diets with seven levels of iron (53.9, 90.0, 115.6, 146.1, 176.0, 215.8 and 266.0 mg iron kg^?1 diet) were fed to Jian carp (initial weight 11.4 ± 0.0 g). Per cent weight gain (PWG), feed efficiency (FE) and protein efficiency ratio were the lowest in fish fed the basal diet (P < 0.05). Body protein content was increased with the increasing iron levels (P < 0.05), but moisture, lipid and ash of fish were not significantly affected by dietary iron levels (P > 0.05). Activities of trypsin, lipase, α‐amylase, Na⁺, K⁺‐ATPase, alkaline phosphatase and gamma‐glutamyl transpeptidase were improved with increasing dietary iron levels. Serum iron were significantly enhanced with dietary iron levels up to 146.1 mg iron kg^?1 diet, and plateaued. In conclusion, iron improved digestive enzyme activities of juvenile Jian carp and the dietary iron requirement for serum iron of juvenile Jian carp (11.4–64.0 g) was 147.4 mg iron kg^?1 diet with ferrous fumarate as the iron source.