葡萄糖和胰岛素对鹅肝细胞脂肪酸合成酶活性及转录表达的影响

通过培养四川白鹅(Anser cygnoides)和朗德鹅(A.anser)原代肝细胞,测定葡萄糖和胰岛素对其甘油三酯(TG)含量,脂肪酸合成酶(FAS)活性及基因表达的影响.结果表明:5 mmol/L葡萄糖或50 nmol/L胰岛素对两种鹅原代肝细胞的TG含量、FAS活性及mRNA表达量影响不显著,而30mmol/L葡萄糖显著增加TG含量、FAS活性及mRNA表达量;5mmol/L葡萄糖与50 nmol/L胰岛素两者共同作用时对TG含量、FAS活性及mRNA表达量诱导效果显著增加,30 mmol/L葡萄糖与50nmol/L胰岛素共同作用的诱导效果更加明显.另外,5 mmol/L葡萄糖与50 nmol/L胰岛素对朗德鹅肝细胞中TG积聚的影响大于四川白鹅,30 mmol/L葡萄糖、5 mmol/L葡萄糖与50 nmol/L胰岛素、30 mmol/L葡萄糖与50 nmol/L胰岛素对朗德鹅FAS基因表达的影响大于四川白鹅.     

油酸对鹅原代肝细胞三酰甘油含量及TNFα和PPA Rα表达的影响

以四川白鹅(Anser cygnoides)原代培养肝细胞为模型,测定了油酸对鹅肝细胞活性、三酰甘油(TG)含量、脂滴形成的影响,同时研究了油酸对肿瘤坏死因子α(TNFα)和过氧化酶体增殖物激活受体α基因(PPARα)表达的作用.结果表明,0.5 mmol/L的油酸能增强肝细胞活性,而1.5 mmol/L油酸对肝细胞活性有抑制作用.添加不同浓度油酸都能诱导鹅肝细胞TG含量和脂滴增多,其中1.0 mmol/L的油酸作用最明显.0.5 mmol/L、1.0 mmol/L和1.5 mmol/L油酸都能提高PPARα mRNA水平;而0.5 mmol/L和1.0 mmol/L油酸均能提高TNFα基因表达,但1.5 mmol/L油酸对TNFα mRNA水平没有明显影响,表明PPARα和TNFα基因表达水平变化对维持鹅肝细胞内脂质代谢的平衡具有重要作用.     

胰岛素对原代培养猪脂肪细胞生脂和脂解相关基因转录表达及脂代谢的影响

为了研究体外胰岛素对猪脂肪组织脂肪代谢及相关基因表达的影响,对取自于7日龄大白×长白杂种仔猪皮下脂肪组织的脂肪细胞,经原代培养,用0!400nmol/L胰岛素处理48h,采用油红O染色提取、甘油试剂盒和半微量RT-PCR分别测定了脂肪细胞生脂和脂解的累计变化,以及转录因子固醇调控元件结合蛋白(SREBP)-1c和碳水化合物反应元件结合蛋白(ChREBP)、脂肪酸合成酶(FAS)、乙酰辅酶A羧化酶(ACC1)和激素敏感脂酶(HSL)基因mRNA水平的变化。结果表明,低糖条件下(5mmol/L)下,胰岛素不影响原代培养猪脂肪细胞ChREBP和ACC1转录表达;胰岛素(200nmol/L例外)明显促进FAS转录表达,100!300nmol/L也显著增强SREBP-1c表达。但二者表达变化不一致,在原代猪脂肪细胞胰岛素是否是通过SREBP-1c途径调控FAS转录尚待进一步研究;高浓度胰岛素(300!400nmol/L)显著促进脂肪细胞HSL表达和脂解活性。     

鸭SCD1基因过表达对原代肝细胞脂质代谢的效应研究

硬脂酰辅酶A去饱和酶1(Stearoy-Co A desaturase 1,SCD1)是肝细胞合成单不饱和脂肪酸的限速酶,对机体脂肪酸的组成具有重要调控作用。为了探究鸭(Anas platyrhynchos)SCD1基因对其脂肪沉积的遗传调控机制,本实验采用Ⅳ型胶原酶消化肝脏分离培养法分离21日鸭胚原代肝细胞并进行培养,构建携带HIS标签的p EGFP-N3-SCD1真核过表达载体,转染鸭胚原代肝细胞,利用HIS标签检测试剂盒测定SCD1基因在鸭胚原代肝细胞中的过表达量,探讨其过表达对鸭原代肝细胞脂质指标的效应。实验结果显示肝细胞初分离效果良好,细胞培养至96 h基本贴满培养板,p EGFP-N3-SCD1重组质粒在肝细胞中能稳定过表达并发出绿色荧光,SCD1基因的过表达与总胆固醇(total cholesterol,TC)及低密度脂蛋白(low density lipoprotein,LDL)含量呈极显著正相关(P0.01),与高密度脂蛋白(high density lipoprotein,HDL)含量呈极显著负相关(P0.01),与甘油三酯(triglyceride,TG)则不相关(P0.05)。结果表明:Ⅳ型胶原酶消化肝脏提取法能成功分离培养鸭原代肝细胞,p EGFP-N3-SCD1重组质粒转染效果良好,SCD1基因过表达对鸭原代肝细胞的脂质代谢具有调控作用,这对今后进一步研究SCD1对肉鸭脂肪沉积的遗传调控机制具有重要的指导意义。     

甘草提取物对叔丁基氢过氧化物(t-BHP)诱导的建鲤原代培养肝细胞损伤的保护作用

以肝(细胞)损伤为主要特征的鱼类肝胆综合症是水产养殖中日趋严重的病害之一,目前还没有有效的防治措施.本研究拟以叔丁基氢过氧化物(t-BHP)构建建鲤(Cyprinus carpio var.jian)原代肝细胞损伤模型,并利用该模型评价甘草(Glycyrrhiza glabra)提取物对t-BHP诱导的鱼类急性肝细胞损伤的保护作用.1 mmol/L的t-BHP与原代肝细胞共培养2h能显著提高肝细胞培养上清中谷丙转氨酶(GPT)、谷草转氨酶(GOT)、乳酸脱氢酶(LDH)和丙二醛(MDA)水平,显著降低谷胱甘肽过氧化物酶(GSH-Px)和超氧化物歧化酶(SOD)的含量以及肝细胞增殖活性.在t-BHP诱导肝细胞损伤前(前处理)、损伤后(后处理)、损伤前和损伤后(前后处理)将不同浓度(0.1、0.2和0.4 mg/mL)的甘草提取物加入肝细胞培养液中,与肝细胞共培养2h,结果显示,前后处理时,不同浓度(0.1、0.2和0.4 mg/mL)的甘草提取物均能显著抑制t-BHP诱导的GOT、GPT、LDH和MDA水平的升高,恢复GSH-Px和SOD水平;前处理时,高浓度(0.4 mg/mL)的甘草提取物对抑制GOT、GPT、LDH和MDA水平的升高,恢复GSH-Px水平有显著效果;后处理时,只有高浓度(0.4 mg/mL)的甘草提取物能有效提高GSH-Px活性;中浓度和高浓度(0.2和0.4mg/mL)的甘草提取物在前处理、后处理及前后处理时均能显著提高肝细胞的增殖活力.研究的结果表明,中药与损伤剂的给予顺序影响着甘草提取物对肝细胞的保护作用,前后处理时甘草提取物对损伤肝细胞的保护效果明显优于前处理和后处理.研究证实了甘草提取物对t-BHP诱导的鱼类肝细胞损伤具有保护作用,对应用甘草提取物作为鱼类肝胆综合症的防治药物还需要进一步的在体研究.     

不同变种甜瓜糖分积累及蔗糖代谢酶活性动态变化

为了解不同类型甜瓜糖分积累及糖代谢特点,选用厚皮甜瓜品种X228、普通甜瓜品种B154及越瓜品种H227为材料,定期取样测定果实成熟过程中的葡萄糖、果糖、蔗糖含量和蔗糖代谢相关酶活性,研究不同变种甜瓜果实发育过程中糖分积累及相关酶动态变化差异。结果表明,授粉15 d至果实成熟期间,3个甜瓜品种的果实葡萄糖与果糖含量的变化均较小,品种间差异不显著。3个甜瓜品种果实蔗糖含量存在显著性差异,其中H227果实几乎无蔗糖积累,葡萄糖和果糖是果实的主要糖组分;B154和X228果实蔗糖含量随着果实发育而快速增加,蔗糖积累存在明显的转折点,蔗糖是B154和X228这2个品种成熟果实中最主要的糖组分,且果实蔗糖含量提高的同时蔗糖磷酸合成酶(SPS)活性上升、酸性转化酶(AI)活性降低,蔗糖合成酶(SS)合成方向的活性与蔗糖含量关系不显著。根据蔗糖含量的差异,可将甜瓜分为蔗糖积累型和低蔗糖积累型两类,前者果实蔗糖含量的上升被认为是SPS活性上升与转化酶(特别是AI)活性下降共同作用的结果,后者果实内极低的蔗糖含量被认为是SPS活性较低导致的。本研究结果为甜瓜种质资源创新利用和甜瓜果实糖分积累调控研究奠定了理论基础。     

HepG2细胞胰岛素抵抗模型的建立及其应用

为建立方便、可靠、重复性好的胰岛素抵抗细胞模型,研究胰岛素受体底物-2(IRS-2)蛋白磷酸化与胰岛素抵抗的关系以及不同铬制剂对细胞葡萄糖代谢的作用。试验采用高胰岛素、高糖联合诱导Hep G2细胞建立胰岛素抵抗模型,通过葡萄糖氧化酶法及噻唑蓝比色法(MTT)分析受试物对细胞葡萄糖代谢及活性的影响,优化最佳造模条件;同时,利用蛋白免疫印迹法(WB)分析IRS-2的蛋白表达及其蛋白磷酸化,验证模型的胰岛素抵抗性。在此基础上,将模型应用于分析葡萄糖耐量因子(GTF)及其它铬制剂的降糖效果。结果表明,高胰岛素(10-6mol·L~(-1))和高糖培养基(25 mmol·L~(-1))处理细胞48h,细胞存活率达96%,与对照组相比,葡萄糖消耗量降低了5.7%,IRS-2含量减少31.02%,胰岛素抵抗效果显著,且该胰岛素抵抗模型可稳定维持48 h。相比于吡啶酸铬和三氯化铬,GTF对细胞葡萄糖代谢有显著的改善作用,其最佳作用浓度为1.0μg·m L-1。利用HepG2细胞建立体外胰岛素抵抗模型,方法简单可靠、重复性好,可广泛应用于天然活性物质的降糖功能研究。     

叶面喷施磷酸二氢钾与葡萄糖对番茄光合速率和蔗糖代谢的影响

以辽园多丽和樱桃番茄红玉为试材，采用叶面喷施磷酸二氢钾、葡萄糖及其两者的混合液，研究叶面喷肥对两个品种番茄光合速率及蔗糖代谢的影响。结果表明：叶面喷肥可明显提高番茄叶片中叶绿素的含量及叶片的净光合速率。通过叶面喷肥处理，提高了2个品种番茄果实内果糖和葡萄糖的含量，提高了果实中酸性转化酶和中性转化酶的活力水平，蔗糖合成酶（SS）和蔗糖磷酸合成酶（SPS）活力也得到明显提高。以叶面喷施磷酸二氢钾及其与葡萄糖两者的混合液效果较好，单独喷施葡萄糖效果稍差。表明在叶面喷施葡萄糖、磷酸二氢钾和两者的混合液条件下，果实是通过提高4种酶的活力来提高库强度的。果实库强度增加的同时，相应地促进了同化产物的运转和干物质的积累。     

皖西白鹅肝脏中GHR，IGF-1和 IGF-1R基因的表达及与朗德鹅的比较

选择90日龄的皖西白鹅和朗德鹅各20只,取肝脏组织。采用半定量逆转录多聚酶链式反应（RT-PCR）的方法,以β-actin为内标,对肝脏组织中生长激素受体（GHR）、胰岛素样生长因子-1（IGF-1）、胰岛素样生长因子-1型受体（IGF-1R）的mRNA表达量分别进行比较分析,并分别与鹅的肝脏重及肝重率进行相关性分析。结果表明：皖西白鹅的肝脏重和肝重率都显著大于朗德鹅（p<0.05）;皖西白鹅肝脏中IGF-1的mRNA表达明显高于朗德鹅（p<0.01）;而GHR、IGF-1R mRNA在两品种间没有显著差异。体重与肝脏重显著正相关（r = 0.35,p<0.05）,肝重率与肝脏中IGF-1的mRNA的表达显著正相关（r = 0.67,p<0.01）,而与GHR、IGF-1R mRNA没有显著的相关性。结果提示：（1）90日龄的皖西白鹅肝脏生长优于朗德鹅;（2）两品种鹅肝脏生长的差异可能与肝脏中IGF-1 mRNA表达的差异有关。     

胰岛素和葡萄糖对水牛体外受精早期胚胎发育的影响

探讨了不同浓度的胰岛素和葡萄糖对水牛体外受精早期胚胎体外发育的影响。结果表明,添加胰岛素浓度为5μg/mL时,水牛早期胚胎的囊胚发育率、囊胚细胞数及冷冻-解冻后胚胎的孵化率都显著高于对照组及其他浓度组(P<0.05);加入葡萄糖的浓度大于6 mm o l/L时显著影响水牛早期胚胎发育到囊胚及孵化囊胚的阶段(P<0.05);联合添加胰岛素和葡萄糖时能显著提高水牛早期胚胎的孵化囊胚率、总囊胚率和囊胚细胞数(P<0.05)。实验证实,浓度为5μg/mL胰岛素能促进水牛体外受精早期胚胎的发育;在TCM-199中单独添加葡萄糖是不必要的,联合添加葡萄糖和胰岛素能促进水牛体外受精早期胚胎的发育。     

保护剂减轻氯嘧磺隆对玉米残留药害的作用机理

采用沙培方法,利用生理生化试验及半定量PCR方法,研究了保护剂HN对玉米ALS、GSTs活性及GSTs的mRNA表达量的影响。结果表明,保护剂对6个玉米品种的ALS、GSTs均具有诱导作用,但对不同品种的诱导增加百分率却不同。对氯嘧磺隆敏感的玉米东甜3号的GSTs诱导增加百分率达到59.18,对其ALS的诱导倍数为叶部1.29倍,根部2.30倍;对氯嘧磺隆耐性较高的玉米屯玉88GSTs诱导增加百分率为19.26,其ALS的诱导倍数为叶部1.07倍,根部2.07倍。氯嘧磺隆和保护剂均可以诱导玉米GSTsmRNA的相对表达量的增加,保护剂的诱导作用更强。     

Identification of benzophenone C-glucosides from mango tree leaves and their inhibitory effect on triglyceride accumulation in 3T3-L1 adipocytes

A 70% ethanol-water extract from the leaves of Mangifera indica L. (Anacardiaceae) inhibited triglyceride (TG) accumulation in 3T3-L1 cells. From the active fraction, seven new benzophenone C-glycosides, foliamangiferosides A (1), A(1) (2), A(2) (3), B (4), C(1) (5), C(2) (6), and C(3) (7), together with five known compounds were isolated and the structures were elucidated on the basis of chemical and physicochemical evidence. The effects of these compounds on TG and the free fatty acid level in 3T3-L1 cells were determined, and the structure-activity relationship was discussed. On the basis of the AMPK signaling pathway, several compounds were found to increase the AMPK enzyme expression and down-regulate lipogenic enzyme gene expression such as SREBP1c, FAS, and HSL.     

重组马白细胞介素-18生物学活性的测定及单克隆抗体中和活性鉴定

应用实时荧光定量RT-PCR方法评价了大肠杆菌表达的重组马白细胞介素-18（rEIL-18）蛋白在商品化的重组人IL-12（rhIL-12）协同作用下刺激马外周血单核细胞（PBMCs）产生EIFN-gamma的情况。在rhIL-12的协同作用下,rEIL-18在体外刺激马PBMCs产生EIFN-gamma的效应是阴性对照的20倍之多,并且在一定rhIL-12浓度的条件下,rEIL-18在体外诱导马PBMCs产生EIFN-gamma的能力是呈一定的剂量依赖性。此外,评价了制备的9株抗rEIL-18单克隆抗体中和rEIL-18的生物学活性的能力,证实其中1株可中和rEIL-18的生物学活性,并且呈一定的剂量依赖性。结果表明：首次获得原核表达的具有生物活性的rEIL-18,其活性可被1株抗rEIL-18单克隆抗体中和。     

Induction of glucokinase mRNA by dietary phenolic compounds in rat liver cells in vitro

Diabetes and its complications, including oxidative stress, are major reasons for medical intervention and one of the most frequent causes of death in developed countries. Several lines of data suggest that the use of certain dietary polyphenolic compounds may alter glucose metabolism, thus decreasing the risk for type 2 diabetes. In this paper, we present the effect of phenolic acids (caffeic, chlorogenic, rosmarinic, and ferulic) and extracts from Smallanthus sonchifolius and Prunella vulgaris on glucose production in rat hepatocytes and on glucokinase, glucose-6-phosphatase, and phosphoenol-pyruvate carboxykinase mRNA expression in rat hepatoma Fao cells. The phenolics at 500 microM and after 1 h incubation lowered glucose production via both gluconeogenesis (10 mM alanine or dihydroxyacetone as precursors) and glycogenolysis compared with metformin. Most of the phenolics increased the level of glucokinase mRNA after 24 h in the same way as insulin (10(-7) M).     

皖西白鹅Pit-1基因插入/缺失多态性及其对早期体重的影响

本文根据鸡Pit-1基因mRNA 序列设计一对引物,以皖西白鹅DNA为模板,扩增鹅Pit-1基因的部分片段,结果发现该片段存在2个插入/缺失突变,共出现3种基因型,分别为AA、AB和BB,与AA型相比BB型分别在扩增片段的132和148bp后插入3 bp和13 bp。卡方检验结果表明,皖西白鹅的基因型分布符合哈代温伯格平衡。不同基因型之间各期体重比较的结果表明,AB型、BB型个体3-11周龄体重均显著高于AA型（P<0.05）;BB型个体3-11周龄体重高于AB型,但差异不显著（P>0.05）,表明就影响鹅早期体重而言,B等位基因为有利基因。     

AGE-induced interference of glucose uptake and transport as a possible cause of insulin resistance in adipocytes

The purpose of this study was to investigate the distinct roles of advanced glycation end products (AGEs) on insulin-mediated glucose disposal in 3T3-L1 adipocytes and C2C12 skeletal muscle cells. AGE-modified proteins, namely, GO-AGEs, were prepared by incubating bovine serum albumin (BSA) with glyoxal (GO) for 7 days. Glucose utilization rates and the expression of insulin signaling-associated proteins, including Akt, insulin receptor substrate-1, and glucose transporter 4, were determined. GO-AGEs caused insulin resistance (IR) by suppressing insulin-stimulated glucose uptake both in 3T3-L1 adipocytes and C2C12 muscle cells. Interestingly, an unexpected finding was that insulin-stimulated glucose transport in adipocytes was affected by GO-AGEs in a biphasic manner, with an initial steep increase (168%) during the first 8 h of incubation followed by a significantly impaired uptake after extended culture times (24-48 h, p < 0.05). Treatment with GO-AGEs for 24 h markedly accelerated lipid droplet formation compared to the BSA control; however, it was blocked by incubation with an anti-RAGE antibody. Our study suggests that GO-AGEs induce an early dramatic elevation of glucose transport in adipocytes that may be related to the activation of insulin signaling; however, subsequent IR may result from increased oxidative stress and proinflammatory TNF-α production.     

Steam-dried ginseng berry fermented with Lactobacillus plantarum controls the increase of blood glucose and body weight in type 2 obese diabetic db/db mice

This study examined whether steam-dried ginseng berries fermented with Lactobacillus plantarum (FSGB) could improve the indices of type 2 diabetes mellitus (T2DM) in obese db/db mice. FSGB was shown to have an effect on body weight and blood glucose/serum parameters when administered at a dose of 0.5 g/kg. In the intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT), FSGB was clearly shown to improve insulin tolerance and glucose tolerance. Moreover, FSGB was shown to enhance immune activities by increasing the immune cell population, and glucose transpoter 1 (GLUT1) mRNA expression in L6 cells was up-regulated, suggesting that FSGB can increase glucose transport activity in target cells. These results indicate that steam- and dry-processed ginseng berries fermented with L. plantarum can be used to effectively control blood sugar metabolism via improving insulin and glucose tolerance and body weight gain in db/db mice.     

Tea polyphenol (-)-epigallocatechin 3-gallate suppresses heregulin-beta1-induced fatty acid synthase expression in human breast cancer cells by inhibiting phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase cascade signaling

Tumor-associated fatty acid synthase (FAS) is implicated in tumorigenesis and connected to HER2 (human epidermal growth factor receptor 2) by systemic analyses. Suppression of FAS in cancer cells may lead to growth inhibition and cell apoptosis. Our previous study demonstrated that (-)-epigallocatechin 3-gallate (EGCG), the green tea catechin, could down-regulate FAS expression by suppressing EGFR (epidermal growth factor receptor) signaling and downstream phosphatidylinositol 3-kinase (PI3K)/Akt activation in the MCF-7 breast cancer cell line. Herein, we examined the effects of EGCG on FAS expression modulated by another member of the erbB family, that is, HER2 or HER3. We identified that heregulin-beta1 (HRG-beta1), a HER3 ligand, stimulated dose-dependent FAS expression in breast cancer cell lines MCF-7 and AU565, but not MDA-MB-453. The time-dependent increase in FAS expression after HRG-beta1 stimulation was also observed in MCF-7 cells, and this up-regulation was de novo RNA synthesis dependent. Treatment of MCF-7 cells with EGCG markedly inhibited HRG-beta1-dependent induction of mRNA and protein of FAS. EGCG also decreased the phosphorylation of Akt and extracellular signal-regulated kinase 1/2 that were demonstrated as selected downstream HRG-beta1-responsive kinases required for FAS expression using dominant-negative Akt, PI3K inhibitors (LY294002 and wortmannin), or MEK inhibitor (PD98059). FAS induction by HRG-beta1 was also blocked by AG825, a selective HER2 inhibitor, and by genistein, a selective tyrosine kinase inhibitor, indicating the formation of a heterodimer between HER2 and HER3, and their tyrosine kinase activities are essential for HRG-beta1-mediated elevation of FAS. Additionally, growth inhibition of HRG-beta1-treated cells was parallel to suppression of FAS by EGCG. Taken together, these findings extend our previous study to indicate that EGCG may be useful in the chemoprevention of breast carcinoma in which FAS overexpression results from HER2 or/and HER3 signaling.