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。     

基于一种新的天花板培养方法分析猪成熟脂肪细胞去分化过程中关键基因表达模式

成熟脂肪细胞在天花板培养(ceiling culture)条件下可自发去分化为不含脂滴的成纤维细胞(dedifferentiated fat cell,DFAT),这种细胞体外可向多个谱系分化,因此近年来备受人们关注。为研究猪成熟脂肪细胞去分化过程中关键基因表达规律,本研究先分离、鉴定了猪成熟脂肪细胞,然后采用一种新的天花板培养法使其发生去分化,再用实时定量PCR(RT-qPCR)技术分析了猪成熟脂肪细胞去分化过程中关键基因的表达变化,最后用1μmol/L罗格列酮(Rosiglitazone)处理猪成熟脂肪细胞,研究增强成脂对去分化的影响。结果显示:分离的猪成熟脂肪细胞的纯度高达98.7%,并且新式天花板培养法能够高效的使其发生去分化;在去分化的过程中中后期成脂关键基因过氧化物酶体增殖物激活受体γ(PPARγ)、脂肪细胞型脂肪酸结合蛋白(aP2)和脂蛋白酯酶(LPL)的mRNA水平分别上调了8、3和7.5倍,而脂肪分解关键基因激素敏感脂酶(HSL)和脂肪组织甘油三酯脂肪酶(ATGL)的mRNA水平分别上调了40倍和10倍;罗格列酮处理能够显著抑制猪成熟脂肪细胞去分化(P<0.05)。这些结果说明,成熟脂肪细胞去分化是一个以脂解为主并伴有一定水平成脂的脂代谢过程,这为进一步研究成熟脂肪细胞去分化机理提供了理论依据。     

过表达miR-128a抑制大鼠前体脂肪细胞分化

为阐明miR-128a在大鼠前体脂肪细胞分化过程中的功能,本实验检测了大鼠(Rattus norvegicus)前体脂肪细胞成脂分化过程中miR-128a的表达,明确其在分化过程中的表达趋势;构建miR-128a的腺病毒过表达载体并侵染大鼠前体脂肪细胞,随后采用Real-time PCR和Western blot分别在mRNA和蛋白水平检测了成脂标记基因过氧化物酶体增生物激活受体γ(peroxisome proliferator-activated receptor gamma,PPARγ)和脂肪酸结合蛋白2(adipocyte protein 2,aP2)的表达;过表达miR-128a的大鼠前体脂肪细胞在第10天进行油红O染色,从形态学上观察成脂分化情况.结果显示,miR-128a的表达量在脂肪细胞诱导分化第二天降到最低点,随后维持在第0天的水平.在过表达miR-128a后,大鼠前体脂肪细胞成脂标记基因PPARγ和aP2的蛋白表达量与对照相比显著下降,脂滴明显少于对照组.实验结果表明:在大鼠前体脂肪细胞中过表达miR-128a能够抑制前体脂肪细胞的分化;并且通过PPARγ mRNA和蛋白水平的差异性变化,结合miRNA的作用机理,推测PPARγ有可能是miR-128a的靶基因.本实验也为研究miR-128a在脂肪细胞分化中发挥作用的机理提供了理论基础.     

猪成熟脂肪细胞分离培养及去分化研究

成熟脂肪去分化技术可为研究脂肪细胞分化提供均一的前体脂肪细胞。本研究分离培养了猪成熟脂肪细胞,并去分化为前体脂肪细胞。本实验采用Ⅱ胶原酶消化后离心分离1~3日龄仔猪(Susscrofa)皮下脂肪组织,天花板法培养获得成熟脂肪细胞。显微镜下观察脂肪细胞去分化形态学变化,并在成脂诱导培养液的作用下诱导再分化。采用油红O染色法检测分化不同时期细胞脂滴聚集效率,脂滴的累积随诱导的进行不断增加。RT-PCR检测成熟脂肪细胞标志基因过氧化物酶体增殖物活化受体(PPARγ)和和脂肪酸结合蛋白4(FABP4)的mRNA相对表达量,分化早期基因表达水平较低,其表达水平在分化过程中持续增高,在分化后期PPARγ相对表达量与诱导分化前增加了2.8倍,FABP4增加了约62倍(差异显著P<0.05)。说明去分化获得的前体脂肪细胞在成脂诱导培养液作用下,可有效地分化为成熟脂肪细胞。本研究优化了猪成熟脂肪细胞分离和培养体系,并通过去分化获得具有再分化能力的前体脂肪细胞,为进一步深入研究猪脂肪细胞分化与代谢提供技术平台。     

猪肌内前体脂肪细胞的体外培养

本研究建立了猪肌内前体脂肪细胞的体外培养模型,以期为更深入研究猪肌内脂肪代谢提供新的实验方法。实验采集出生5d仔猪的背最长肌组织,Ⅱ型胶原酶消化2h后400目筛网过滤,然后1500r/min离心,沉淀用含10%胎牛血清的DMEM/F12培养基(完全培养基)重悬后差速贴壁2h,弃去原有培养基,加入新的含10%胎牛血清的DMEM/F12培养基(完全培养基)进行培养。细胞经传代且完全融合48h后,在完全培养基中添加0.5mmol/L3-异丁基-1-甲基黄嘌呤(IBMX),1μmol/L地塞米松(DEX),10mg/L胰岛素(INS)进行诱导培养48h,再换以含10mg/L胰岛素的完全培养基培养48h,最后换完全培养基继续培养,直至90%的细胞出现脂滴。培养结果:细胞贴壁生长,呈短梭状或棱形,经诱导培养后细胞充脂率高。经形态学观察、生长曲线及油红O脂肪染色法鉴定,证明是肌内前体脂肪细胞。普通PCR及实时荧光定量PCR均检测到了脂联素基因的表达。本研究通过差速贴壁法成功培养了猪肌内前体脂肪细胞,并通过诱导培养重现了其分化、成熟的全过程。     

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.     

慢病毒介导shRNA干扰颗粒体蛋白前体(GRN)促进猪前体脂肪细胞分化

为研究颗粒体蛋白前体(granulin,GRN)在脂肪细胞发育过程中的作用,本实验构建了GRN短发夹RNA(short hairpin RNA,shRNA)慢病毒干扰载体,包装并感染猪前体脂肪细胞,采用油红O染色、油红O提取比色法检测猪前体脂肪细胞分化情况,采用Real-time PCR法检测成脂关键基因mRNA的表达变化情况。结果显示,GRN慢病毒干扰载体病毒滴度在5×107TU/mL以上,病毒感染前体脂肪细胞后显著降低了GRN的表达,shRNA2干扰效率最高,达到76%,沉默GRN后能够促进猪前体脂肪细胞分化;脂肪细胞分化标志基因过氧化物酶体增殖物激活受体γ(PPARγ)、固醇调节原件结合蛋白(SREBP-1c)、脂肪细胞型脂肪酸结合蛋白(ap2)和脂蛋白酯酶(LPL)基因mRNA表达量均升高。结果表明,降低GRN基因表达促进了猪前体脂肪细胞分化,揭示GRN在猪前体脂肪细胞分化过程中可能起到抑制作用。     

雷帕霉素靶蛋白(mTOR)信号通路参与沉默信息调节因子1(Sirt1)抑制小鼠脂肪沉积

探讨沉默信息调节因子1(Sirt1)对小鼠脂肪沉积的抑制作用和雷帕霉素靶蛋白(mTOR)信号通路的影响。用Sirt1的激动剂白藜芦醇(100mg·kg-·1d-1)和抑制剂尼克酰胺(500mg·kg-·1d-1)灌胃处理小鼠(Mus mussulus)15d,记录小鼠体质量变化,测定小鼠皮下脂肪、附睾脂肪和肾周脂肪的沉积量,试剂盒测定血脂指标,同时利用Real-timePCR分析与脂肪生成密切相关的转录因子过氧化物酶体增殖物激活受体γ(PPARγ)、固醇调节元件结合蛋白1(SREBP1)和脂解基因甘油三酯水解酶(ATGL)、激素敏感性脂肪酶(HSL)、围脂滴蛋白(Perilipin)及生脂基因脂肪酸合成酶(FAS)mRNA的表达水平,检测mTOR通路关键因子雷帕霉素靶蛋白(mTOR)、核糖体S6蛋白激酶1(S6K1)和真核启动因子4E结合蛋白1(4EBP1)mRNA表达水平。与对照组相比,白藜芦醇处理组小鼠体质量增加量和体脂含量均显著降低(P<0.01),血清中甘油三酯(TG)、总胆固醇(TC)和低密度脂蛋白(LDL-C)浓度均显著降低(P<0.01),而高密度脂蛋白(HDL-C)浓度升高(P<0.01),mTOR通路关键因子mTOR、4EBP1和S6K1mRNA表达水平降低(P<0.01),脂代谢相关基因PPARγ、SREBP1及成脂基因FASmRNA表达水平显著降低(P<0.01),脂解相关基因ATGL、HSL和PerilipinmRNA表达水平显著升高(P<0.01);烟酰胺处理组小鼠体质量、附睾脂肪以与皮下脂肪沉积量增加缓慢(P>0.05),肾周脂肪沉积量增加(P<0.05),血清中LDL-C浓度升高(P<0.05),HDL-C浓度降低(P<0.01),mTOR通路关键因子mTOR和4EBP1mRNA表达水平升高(P<0.01),而脂代谢调控相关因子PPARγ和SREBP1mRNA水平升高(P<0.05),ATGLmRNA表达量显著降低(P<0.05),FAS、HSL和PerilipinmRNA表达量变化不显著(P>0.05)。表明激活Sirt1可减少脂肪合成,增加脂肪分解,从而降低体脂沉积,而mTOR信号通路参与这个过程。     

日粮共轭亚油酸对黄羽肉鸡腹脂沉积的影响

选择1日龄黄羽肉鸡60只,随机分成2组。在实验组中添加3%共轭亚油酸（conjugated linoleic acid,CLA）,对照组日粮中添加3%的食用菜籽油。49日龄屠宰,采集腹部脂肪组织。用RT-PCR方法,以β-actin为内标,相对定量测定腹脂中鸡生长激素受体（cGHR）、胰岛素样生长因子-1（cIGF-1）、胰岛素样生长因子Ⅰ型受体（cIGF-ⅠR）、过氧化物增殖剂活化受体γ（cP-PARγ）、脂联素（cAdiponectin）及其Ⅰ型受体（cAdipoⅠR）的mRNA丰度。结果显示,CLA处理使肉鸡的腹脂沉积下降了20.93%（P〈0.05）,分别使腹脂cGHR mRNA和cPPARγmRNA降低了24.74%（P〈0.05）和66.52%（P〈0.01）;而对cIGF-1、cIGF-ⅠR、cAdiponectin和cAdipoⅠR基因表达无显著影响（P〉0.05）;cGHR和cPPARγmRNA的表达与腹脂率正相关（P〈0.05）。数据说明,CLA降低肉鸡脂肪沉积的作用可能通过降低GHR和PPARγmRNA水平实现,而与IGF-1和（或）GH-IGF-1途径、Adiponectin的作用无关。     

Effect of long-term application of rice straw,farmyard manure and inorganic fertilizer on potassium dynamics in soil

Knowledge of K-dynamics in soils can help devise practices for efficient K management in intensive rice-wheat systems. We studied the effect of long-term application of rice straw, farmyard manure (FYM) and inorganic fertilizer on total K and its distribution among different forms in 60-cm soil profile after 14 years of rice-wheat cropping. The exchangeable, the non-exchangeable and the lattice K respectively comprised 1%, 3–10% and 89–95% of total K in surface soil under different treatments. Application of rice straw and FYM positively impacted total K status of soil and its distribution among different forms. The greatest concentrations of total K, lattice K, exchangeable K and NH₄OAc-extractable K were observed in plots receiving both rice straw and FYM together and the lowest in inorganic fertilizer treated plots. On the contrary, the non-exchangeable K was the highest in inorganically fertilized plots and the lowest in rice straw amended plots. The exchangeable, the water soluble and the NH₄OAc-extractable K decreased with soil depth and did not indicate K movement beyond the rooting zone of the crops. The results showed that incorporation of rice residue in soil, instead of burning, besides reducing environmental pollution led to improved K-fertility of soils.     

Potassium Dynamics in Inceptisols as Influenced by Graded Levels of Potash for Banana: I. Potassium Fractions

Knowledge of forms of potassium (K) in soil is of great importance for formulating sound fertilizer recommendation to banana. Field experiments were conducted to study the effect of graded levels of potash application on forms of potassium at juvenile, grand growth, shooting, and harvest stages of banana on Vertic Haplustept soil. The negative balance of soil-available K was observed in the treatments of 0, 100, and 200 g K₂O plant^?1. However, at 300-g K₂O plant^?1 level, the balance was a mere –11 kg ha^?1, and positive balance was observed in the treatments of 400 and 500 g K₂O plant^?1. Significant positive correlation among different forms of K indicated the interdependency and dynamic equilibrium between K forms. Application of K₂O above the level of 300 g plant^?1 for banana resulted in the build-up of potassium fractions in soil.     

Potassium release by some delaware soils

Abstract

The A and B horizons of 29 Delaware soils were extensively cropped to sudan grass (Sorghum vulgare Pers. Piper) and corn (Zea mays L. Pioneer Brand Hybrid No. 3958) to evaluate the K release characteristics of these soils.

Various chemical analyses were made on these soils. The double acid North Carolina extractant (NCE) was good at predicting K uptake during the early stages of the experiment and was most sensitive with A horizon soils. However, this extractant was not suitable for predicting K uptake in the latter part of the experiment when nonexchangeable K was more important. Boiling IN HNO₃ was a good extractant in the latter part of the experiment when nonexchangeable K was being released and it also predicted K uptake from the B horizon well. Soils leached with 0.025M MgCl₂ and incubated 25 days to determine K released were also highly correlated with plant uptake.     

共施磷酸二氢钙和硫酸铵对土壤中钾形态转化的影响

Soil potassium (K) deficiency has been increasing over recent decades as a result of higher inputs of N and P fertilizers concomitant with lower inputs of K fertilizers in China; however, the effects of interactions between N, P, and K of fertilizers on K status in soils have not been thoroughly investigated for optimizing N, P, and K fertilizer use effciency. The influence of ammonium sulfate (AS), monocalcium phosphate (MCP), and potassium chloride application on K fractions in three typical soils of China was evaluated during 90-d laboratory soil incubation. The presence of AS significantly altered the distribution of native and added K in soils, while addition of MCP did not significantly affected K equilibrium in most cases. Addition of AS significantly increased water-soluble K (WSK), decreased exchangeable K (EK) in almost all the soils except the paddy soil that contained considerable amounts of 2:1 type clay minerals with K added, retarded the formation of fixed K in the soils with K added, and suppressed the release of fixed K in the three soils without K added. These interactions might be expected to influence the K availability to plants when the soil was fertilized with AS. To improve K fertilizer use effciency, whether combined application of AS and K was to be recommended or avoided should depend on K status of the soil, soil properties, and cropping systems.     

小麦-玉米轮作系统下长期施钾对作物产量、钾素利用率和钾素平衡的影响

Sustainable potassium (K) management at different soil sites requires understanding the relationships between crop productivity and long-term K fertilizations on a regional or national scale.We analyzed responses of grain yield of wheat (Triticum aestivum L.) and maize (Zea mays L.),K efficiency,and partial balance (difference between K input through fertilizer and K output in the aboveground biomass) during 15-(1990-2005) or 18-year (1990-2008) K fertilizations at five distinctive agroecological zones across China.Compared to the inorganic nitrogen (N) and phosphorus (P) fertilization,the inorganic NPK fertilization significantly increased grain yields of wheat (21%) and maize (16%-72%) at Qiyang and Changping,where soils have low exchangeable and non-exchangeable K contents,but not at rmqi,Yangling and Zhengzhou,where soils have a high exchangeable and non-exchangeable K and/or low N/K ratio in crop plants.Compared to the inorganic NPK fertilization,the inorganic NPK (30% N) and organic manure (70% N) fertilization (NPKM) increased grain yields of wheat (14%-40%) and maize (9%-61%) at four sites,but not at Zhengzhou.For a productivity of wheat at 2-5 t ha-1 or maize at 3-6 t ha-1,13-26 or 9-17 kg K ha-1 were required to produce 1.0 t wheat or maize.The NP fertilization resulted in the lowest negative partial K balance and accumulated 52 kg K ha-1 year-1 less than the NPK fertilization,which accumulated 28 kg ha-1 year-1 less K than the NPKM fertilization.A re-evaluation of the site-specific fertilization effects on N/K ratio in crop plants and soil K accumulation under current NPK and NPKM fertilization is urgently needed to increase both crop yield and K use efficiency at different agroecological zones across China.     

土壤非交换态钾与结构态钾能够区分吗？

Nonexchangeable K (NEK) is the major portion of the reserve of available K in soil and a primary factor in determining soil K fertility. The questions of how much NEK is in soils and how to quantify total NEK in soils are so far still unclear due to the complicated effects of various minerals on K fixation. In this study, the NEK in 9 soils was extracted with sodium tetraphenylboron (NaBPh₄) for various time periods longer than 1 d. The results showed that the NEK extracted by NaBPh₄ gradually increased with time, but showed no more increase after the duration of extraction exceeded 10--20 d. As the temperature increased from 25 to 45 ^oC, the duration to obtain the maximum extraction of NEK was reduced from 20 to 10 d, and the maximum values of NEK released at both temperatures was almost the same for each soil. The maximum NEK (MNEK) of the 9 soils extracted by NaBPh4 varied from 3 074 to 10 081 mg kg^-1, accounting for 21%--56% of the total soil K. There was no significant correlation between MNEK released by NaBPh₄ and other forms of K, such as NH₄OAc-extracted K, HNO₃-extracted K and total K in soils, which indicates that NEK is a special form of K that has no inevitable relationship to the other forms of K in soils. The MNEK extraction by NaBPh₄ in this study indicated that the total NEK in the soils could be differentiated from soil structural K and quantified with the modified NaBPh₄ method. The high MNEK in soils made NEK much more important in the role of the plant-available K pool. How to fractionate NEK into different fractions and establish the methods to quantify each NEK fraction according to their bioavailability is of great importance for future research.     

Yield of corn as affected by potassium on a coastal plain soil

Abstract

Corn (Zea mays L.) yields were determined over a four year period on Kalmia sandy loam soil at Georgetown, Delaware. Yields were not significantly increased by the application of K fertilizer. Potassium soil test level was not significantly correlated with corn yield. Multiple extraction and leaching and changes in soil test K indicated that this soil has a K equilibrium which is reestablished sufficiently rapid to supply K to a growing corn crop.     

Potassium Indexing of Crops Grown on Eight Benchmark Soil Series of India

Surface soil (0–15 cm) and foliar samples of wheat, rice, sugarcane, and cotton crops growing in eight intensively cultivated soil series of India were collected from farmers' fields during 2004 and 2005. Analysis of soil and leaf samples indicated widespread potassium (K) deficiency in soils and crops. Potassium deficiency in wheat growing in Lukhi series (Udic Ustocrept) and Akbarpur series (Udic Haplustulf) and in cotton growing in Typic Torrifluvent were comparable to 1N ammonium acetate (NH₄OAc)–K (available K) soil test values. Rice cultivated on Udic Ustocrept soils in summer monsoon season showed relatively less K deficiency in comparison to winter wheat, and K deficiency in growing rice did not match 1N NH₄OAc‐K soil test values. In the Khatki series, K deficiency in sugarcane was much less than 1N NH₄OAc‐K soil test values. In the Arsia series (Typic Haplustert), despite the high 1N NH₄OAc‐K status of soils, K deficiency in growing wheat was observed.     

UTX与JMJD3体外转录载体构建及其在小鼠卵母细胞上的验证

UTX(ubiquitously transcribed tetratricopeptide repeat X chromosome)和JMJD3(jumonji domain containing 3)是组蛋白H3赖氨酸27位三甲基化(histone H3 lysine 27 trimethylation,H3K27me3)去甲基化酶,可以调控动物机体生长、肌肉发育、调节代谢等重要生理功能.本研究根据小鼠(Mus musculus)UTX和JMJD3 mRNA序列设计3对引物,分别构建了体外转录载体pMD19T-T7UTX和pMD19T-T7JMJD3.分别将体外转录mRNA注射进小鼠卵母细胞,并利用免疫荧光方法对H3K27me3进行检测.转录产物经电泳检测,结果显示,UTX与JMJD3条带清晰与预期大小相符;通过显微注射mRNA的卵母细胞,其H3K27三甲基化修饰明显减少.本研究成功构建了UTX与JMJD3体外转录载体,并在细胞水平证明转录产物能够翻译出具有活性的酶,为利用此载体研究UTX与JMJD3在生长发育及相关疾病发生中的机制提供基础资料.     

Long-term Potassium Contribution of Potassium Forms of Udic Haplustept and Sustainability of Growing Crops

A balance sheet of potassium (K) was prepared in a long-term experiment that was started in 1985 on a loamy sand Udic Haplustept at the Potash Research Institute of India, Gurgaon, Haryana, India, involving a sorghum (fodder)–wheat cropping system. During the 20 cycles of sorghum and wheat, as much as 2,404 kg K ha^?1 was released from the structural form of soil K that contributed 77.3% K to the total K removed by growing sorghum and wheat. However, this K released from the structural form could no longer sustain the growth of sorghum and wheat. Both water-soluble and exchangeable soil K forms contributed 3% K and nonexchangeable K contributed 6.6% K. Groundwater used in irrigation contributed 13.1% K. Potassium fertilizer is needed for the growth of sorghum and wheat on a loamy sand Udic Haplustept as in this long-term experiment and other representative soils both to sustain yield and maintain soil K fertility.