猪SFRP4基因的表达规律及sp600125对前体脂肪细胞分化的影响

分泌型卷曲相关蛋白（SFRP4, secreted frizzled-related protein 4）是Wnt信号通路可溶解的调控子.本研究通过高通量测序（Solexa）技术、实时定量PCR（RT-qPCR）对瘦肉型和脂肪型猪不同生长阶段脂肪组织中SFRP4表达规律进行研究;用western免疫印迹及RT-PCR技术对脂肪细胞分化过程中SFRP4蛋白表达和mRNA表达进行检测;用JNK信号通路特异性抑制剂sp600125处理猪原代前体脂肪细胞,研究抑制JNK信号通路对猪前体脂肪分化以及SFRP4 mRNA和蛋白表达的影响.结果显示,SFRP4 在脂肪型猪脂肪组织表达量显著高于瘦肉型猪(P<0.01);不同组织检测结果发现,SFRP4广泛表达于各个组织,并高表达于脂肪组织;前体脂肪细胞向成熟脂肪细胞分化过程中SFRP4表达量逐渐升高;sp600125促进了前体脂肪细胞分化,引起了 PPARγ、FABP4 、ATGL、Perilipin的显著升高(P<0.01),而SFRP4的表达被显著抑制.本研究为调控脂肪细胞分化关键基因的筛选提供新的理论参考.     

维生素C通过调控脂肪形成相关基因的转录促进猪前体脂肪细胞的增殖与分化

探讨维生素C（Vit C）诱导猪前体脂肪细胞增殖分化最佳浓度及在分化过程中,5种脂肪形成相关基因peroxisome proliferator activated receptor gamma(PPARγ)和retinoid X receptor alpha(RXRα),脂肪细胞分化标志基因lipoprotein lipase (LPL),生脂基因phosphoenolpyruvate carboxykinase(PEPCK)、stearoyl CoA desaturase(SCD) mRNA表达时序性的变化. 以3　d龄猪前体脂肪细胞为实验对象,用Vit C诱导猪前体脂肪细胞增殖分化,分别在增殖分化第2、4、6和8 d收获细胞,利用MTT测定其增殖程度;油红O染色提取法检测其脂肪含量;采用SQ RT PCR法检测脂肪生成相关基因PPARγ、RXRα、LPL、PEPCK和SCD mRNA表达的变化. 结果显示,PPARγ mRNA在诱导分化第2 d时有低水平表达,在诱导分化过程中表达量逐步升高,在终末分化阶段仍保持高水平表达;RXRα mRNA在诱导分化第2和4 d表达量很低,诱导分化第6 d时表达增加.在诱导分化第8 d,RXRα mRNA表达与第6 d相比差异不显著,直至终末分化. 脂肪细胞分化标志基因LPL在第2 d开始表达,第4和6 d逐步升高,在终末分化阶段仍保持高水平的表达;生脂基因PEPCK和SCD mRNA在第2和4 d开始表达,第6和8 d仍保持高水平的表达. 研究结果表明,100 μmol/L的Vit C促进猪前体脂肪细胞增殖能力最强;250 μmol/L Vit C能显著促进猪前体脂肪细胞分化. 其作用机制可能是通过对转录因子PPARγ和RXRα及标志基因LPL mRNA时序性表达的调控来进行的,促进生脂基因的表达,从而诱导脂肪细胞的分化.     

Role of Phosphotyrosine Interaction Domain Containing 1 in Porcine Intramuscular Preadipocyte Proliferation and Differentiation

Phosphotyrosine interaction domain containing 1 (PID1), a recently identified gene involved in obesity-associated insulin resistance, plays an important role in fat deposition. However, its effect on porcine intramuscular preadipocyte proliferation and differentiation remains poorly understood. In this study, the plasmid pcDNA3.1(+)-pPID1 was transfected into porcine intramuscular preadipocytes with Lipofectamine 3000 reagent to over-express porcine PID1 (pPID1). Over-expression of pPID1 significantly promoted porcine intramuscular preadipocyte proliferation. Expression of pPID1 mRNA was significantly increased upon porcine intramuscular preadipocyte differentiation. Indirect fluorescent immunocytochemistry demonstrated that pPID1 protein was localized predominantly in the nucleus of porcine intramuscular preadipocyte. The mRNA levels of peroxisome proliferators-activated receptor γ, CCAAT/enhancer binding protein α and lipoprotein lipase were significantly increased by pPID1 over-expression. Over-expression of pPID1 also led to an increase in lipid accumulation which was detected by Oil Red O staining, and significantly increased the intramuscular triacylglycerol content. These results indicate that pPID1 may play a role in enhancing porcine intramuscular preadipocyte proliferation and differentiation.     

猪TCTP基因的表达规律及其对脂肪细胞分化的影响

翻译控制肿瘤蛋白（TCTP, translationally controlled tumor protein）是一类广泛存在各种生物、序列高度保守的蛋白,最初认为TCTP是一类生长相关蛋白,近年研究发现TCTP可能具有非常重要的生物学功能.本研究通过高通量测序(Solexa)技术、实时定量PCR (RT qPCR)对瘦肉型和脂肪型猪不同生长阶段脂肪组织、脂肪细胞中TCTP的表达规律进行了研究,采用siRNA技术,沉默TCTP,研究了其对脂肪细胞分化的影响.结果表明：TCTP在瘦肉型猪脂肪组织中的mRNA表达量显著高于脂肪型猪（P＜0.01）|在不同日龄猪脂肪组织中,TCTP的mRNA表达量随着日龄增长而降低|在不同组织中的检测结果发现,TCTP在心、肝、肾、肌肉和脂肪中有较高的表达,肺和脾中表达量较低|TCTP的mRNA表达量在猪前体脂肪细胞增殖过程中逐渐增高,在分化阶段逐渐下降|沉默TCTP促进了脂肪细胞的分化,引起了PPARγ、C/EBPα、SREBP 1c的显著升高（P ＜0.01）.以上研究发现,TCTP在脂肪沉积过程中可能具有抑制作用,为进一步研究肥胖关键基因调控机制提供科学依据.     

Txnip基因siRNA慢病毒表达质粒构建及促进猪前体脂肪细胞分化

硫氧还蛋白互作蛋白(thioredoxin interacting protein, Txnip)是一种氧化还原调节蛋白质,与硫氧还蛋白结合并抑制其活性,调节细胞氧化还原状态,影响细胞多种生理过程,然而其在猪脂肪细胞分化中的作用尚不明确。本文设计合成3对靶向猪Txnip基因的shRNA寡核苷酸,分别连接于重组慢病毒载体pGLV_3/H_1/GFP+Puro构建siRNA表达质粒。测序验证后,与包装质粒共转染293T细胞,获得滴度1×10~8 pfu/mL的慢病毒干扰质粒。以MOI值100转染原代培养猪前体脂肪细胞,转染率均达80%以上,其中Txnip-shRNA-2转染细胞Txnip基因沉默率达75%。转染Txnip-shRNA-2的猪前体脂肪细胞用成脂分化培养液诱导后,每隔1 d检测细胞成脂分化及相关基因表达。结果发现,其分化比阴性对照质粒转染或未转染细胞显著增强(P<0.05),PPARγ和FAS mRNA表达水平显著提高(P<0.05)。本文构建siRNA慢病毒表达质粒能有效干扰猪Txnip基因表达,Txnip表达沉默可通过上调PPARγ表达促进猪前体脂肪细胞分化。本研究提示,Txnip可能是猪脂肪细胞分化的抑制因子。     

Up-regulated miR-145 Expression Inhibits Porcine Preadipocytes Differentiation by Targeting IRS1

Generally, most miRNAs that were up-regulated during differentiation promoted adipogenesis, but our research indicated that up-regulation of miR-145 in porcine preadipocytes did not promote but inhibit adipogenesis. In this study, miR-145 was significantly up-regulated during porcine dedifferentiated fat (DFAT) cells differentiation. In miR-145 overexpressed DFAT cells, adipogenesis was inhibited and triglycerides accumulation was decreased after hormone stimulation (P<0.05). Furthermore, up-regulation of miR-145 expression repressed induction of mRNA levels of adipogenic markers, such as CCAAT/enhancer-binding protein α (C/EBPα), and peroxisome proliferator-activated receptor γ2 (PPARγ2). These effects caused by miR-145 overexpression were mediated by Insulin receptor substrate 1 (IRS1) as a mechanism. These data suggested that induced miR-145 expression during differentiation could inhibit adipogenesis by targeting IRS1, and miR-145 may be novel agent for adipose tissue engineering.     

慢病毒载体介导的RNA干扰Akt2表达抑制猪前体脂肪细胞分化

丝氨酸/苏氨酸蛋白激酶2(serine/threonine protein kinase2,Akt 2)是胰岛素信 号通路的关键基因, 与细胞生存和癌症的发生密切相关. 但Akt2在前体脂肪细胞分化中 的作用仍然不是十分清楚. 本研究构建了慢病毒干扰载体pLentiH1-Akt2-shRNA 1, 2, 3及scrambled, 经酶切和测序鉴定均正确; 这4种干扰载体分别转染HEK293T细胞后, 均 获得有感染性的病毒颗粒并感染猪前体脂肪细胞. 转染HEK293T细胞48 h 后real-time PCR分析和转染72 h 后Western 印迹分析表明, Akt2-shRNA2 和shRNA3 介导的Akt2 mRNA和蛋白表达被显著下调 (P <0.05), 其中pLentiH1-Akt2-shRNA3 介导的对Akt2 mRNA和蛋白的干扰效率均达到70%以上 (P <0.01). 进一步研究发现, 猪前体脂肪细胞 中Akt2被有效干扰后, 细胞中脂滴明显减少并变小, 且成脂标志基因PPARγ和aP2蛋白 水平被显著下调. 本研究结果表明, Akt2 knockdown可显著抑制猪前体脂肪细胞分化.     

白藜芦醇对猪原代前体脂肪细胞的增殖与分化及Sirt1基因转录表达时序的影响

分别以0μmol/L(对照组)、10μmol/L(低剂量组),20μmol/L(中等剂量组),50μmol/L,100μmol/L(高剂量组)的白藜芦醇(Resveratrol,RES)处理体外培养1~3日龄健康仔猪前体脂肪细胞,采用MTT比色法检测细胞活性及增殖状况;油红O染色化学比色法定量分析细胞内脂肪生成及细胞分化程度;RT-PCR法分析Sirt1(sirtuin1)mRNA表达情况,探讨Sirt1对猪前体脂肪细胞增殖分化的影响及其分子机制。结果表明,脂肪细胞经RES处理后,各组MTT和油红O染色测得的光密度值(OD值)均低于对照组,50μmol/L,100μmol/L组在96~120h作用极显著(P<0.01),与中低剂量组差异显著(P<0.05);以20μmol/L,100μmol/LRES处理细胞后,Sirt1mRNA表达量随细胞分化的进行而逐渐升高,100μmol/L组均显著高于对照组和20μmol/L组(P<0.05)。RES对猪前体脂肪细胞增殖分化均有一定抑制作用,高剂量RES(50μmol/L和100μmol/L)可显著减少细胞内脂肪的合成、抑制脂肪细胞增殖与分化,Sirt1mRNA表达量显著升高可能是RES抑制细胞分化的重要原因之一。     

间充质干细胞对造血干/祖细胞分化为巨核细胞的影响

目的:探讨间充质干细胞(MSC)共培养对体外诱导脐带血单个核细胞来源的造血干/祖细胞生成巨核细胞的影响。方法:分离得到骨髓和脐带2种来源的MSC,并对它们进行表面标志和多向分化能力的鉴定,同时通过实时定量PCR及对RT-PCR产物的电泳分析,对比相同培养代数下2种MSC表达造血因子的情况;用梯度离心法分离得到单个核细胞,通过直接接触或Trans-well分隔的方式分别与MSC共培养,观察细胞增殖情况,并检测巨核系特异性的表面标志和相关基因的表达。结果:骨髓和脐带来源的MSC均分泌对巨核细胞增殖分化有促进作用的造血因子,与造血干/祖细胞直接共培养,对于巨核细胞的增殖有明显的促进作用,分化效果不明显;在非接触共培养的条件下,对巨核细胞的增殖及分化都产生促进作用,且骨髓来源的MSC较脐带来源的MSC效果更加明显。结论:MSC与脐带血造血干/祖细胞非接触培养,对其向巨核分化和增殖的促进作用明显,本实验所用的骨髓来源MSC促分化效果更好。本研究为今后进一步优化巨核系诱导分化体系奠定了基础,并对未来体外大规模制备巨核系祖细胞应用于临床治疗有一定的指导作用。     

花生四烯酸对大鼠前体脂肪细胞生长与分化的影响(英文)

以大鼠前体脂肪细胞原代单层培养为模型,用不同浓度花生四烯酸(AA)处理细胞.通过台盼蓝排斥试验及噻唑蓝比色法(MTT)反映各组细胞增殖状况;Hoechst33342荧光染色观察AA处理后细胞核形态变化;油红O染色提取法分析细胞分化程度;逆转录聚合酶链反应(RTPCR)分析环氧合酶2(COX2)mRNA表达情况,探讨外源性AA对大鼠前体脂肪细胞生长分化的影响及其可能机制.120μmolLAA处理前体脂肪细胞24～72h,细胞活力明显高于对照组;160μmolLAA作用48h时,前体脂肪细胞表现出明显的凋亡现象;脂肪细胞经40～80μmolLAA作用72h时,细胞油红O染色的吸光度值显著减少;40μmolLAA在作用的24h时,可显著上调COX2mRNA的表达量.说明外源性AA以时间性和剂量依赖性调节前体脂肪细胞的生长与分化,40～80μmolLAA在不显著增加脂肪数目的同时,可抑制前体脂肪细胞向成熟脂肪细胞转化、减少脂肪生成量,对控制动物体脂的形成有一定参考价值,COX2mRNA表达量的上升可能是AA抑制前体脂肪细胞分化的内在机制.     

过表达RBP4抑制体外培养的猪前体脂肪细胞分化

为研究视黄醇结合蛋白4(retinol binding protein 4,RBP4)对猪前体脂肪细胞分化的影响,实验构建了RBP4重组腺病毒表达载体,包装并感染猪前体细胞,采用油红O染色和Real-time PCR等方法,检测了过表达RBP4对成脂分化的作用. 研究结果显示,重组腺病毒RBP4载体构建成功,转染猪前体脂肪细胞后,使RBP4的mRNA水平和蛋白水平分别增加了约400倍和20倍. 过表达RBP4能减少脂肪细胞的脂质积累,降低成脂关键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. 结果表明,RBP4对猪前体脂肪细胞分化有抑制作用,为进一步研究RBP4对猪前体脂肪细胞分化的作用机制奠定基础.     

Adipocyte Development is Dependent Upon Stem Cell Recruitment and Proliferation of Preadipocytes

KRAS, KRYSTYNA M., DOROTHY B. HAUSMAN, GARY J. HAUSMAN, AND ROY J. MARTIN. Adipocyte development is dependent upon stem cell recruitment and proliferation of preadipocytes. Obes Res. Objectives: The ability to acquire fat cells persists over the life spans of animals. It is unknown whether adipocyte acquisition is the result of preadipocyte proliferation or stem cell recruitment to become adipocytes. The purposes of these studies were 1) to characterize early differentiation of stromal vascular (S-V) cells to preadipocytes as it is influenced by insulin, dexamethasone (DEX), and insulin-like growth factor-I (IGF-I); and 2) to determine whether new fat cells arise from stem cell recruitment or preadipocyte proliferation. Research Methods and Procedures: Freshly isolated S-V cells from rat inguinal adipose tissues were plated for 24 hours then exposed to serum-free medium. Results: Approximately 15% of freshly plated S-V cells were preadipocytes as determined by a preadipocyte specific marker, AD3. Total cell number and proportion of preadipocytes were significantly greater with 100 nM insulin treatment than with 0, 0. 1, or 1. 0 nM, but IGF-I treatment at 10 nM resulted in preadipocyte development similar to that with 100 nM insulin treatment. The addition of 5 nM DEX to the 100 nM insulin treatment resulted in a 20% increase in preadipocyte number by day 2 when compared to either treatment alone. 5-Bromo-2′-deoxyuridine treatment suppressed the increased proportion of preadipocytes from days 0–2 in non-insulin treated cells and prevented the increase typically observed with insulin. A mitosis inhibitor also significantly reduced the proportion of preadipocytes. Discussion: These results show for the first time that S-V cells are recruited as preadipocytes and that proliferation of these preadipocytes and early differentiation occur simultaneously.     

Effect of Acetylcholine Precursors on Proliferation and Differentiation of Astroglial Cells in Primary Cultures

Effects of acetylcholine and of the cholinergic precursors choline, cytidine 5′-diphosphocholine (CDP-choline) and α-glyceril-phosphorylcholine (α-GPC) on transglutaminase (TG) and cyclin D1 expression were studied in primary astrocyte cultures by confocal laser microscopy (CLSM) with monodansyl-cadaverine uptake as a marker of enzyme activity and by immunochemistry (Western blotting). CLSM analysis showed an increased cytofluorescence in 0.1 μM choline-treated astrocytes. Treatment with CDP-choline dose-dependently increased TG. A total of 1 μM CDP-choline exposure in 14 days in vitro (DIV) astrocyte cultures increased cytofluorescence. A total of 1 μM α-GPC 24 h-treated cultures revealed increased cytofluorescence both in cytosol and nuclei. Western blot analysis showed an increased TG expression in cultures exposed for 24 h to 1 μM choline or α-GPC, whereas in 24 h 1 μM CDP-choline and acetylcholine-treated astrocytes TG expression was unaffected. Treatment with 1 μM acetylcholine reduced TG expression at 21 DIV. In cultures at 14 and 35 DIV cholinergic precursor treatment for 24 h induced a marked down-regulation of cyclin D1 expression, with reduced cyclin D1 expression in 1 μM α-GPC treated astrocytes. Our data suggest a role of cholinergic precursors investigated independent from acetylcholine on maturation and differentiation of astroglial cells in vitro, rather than on their growth, proliferation and development in culture. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

磷离子对BM-hMSCs增殖及成骨分化的影响

目的:探讨磷离子对人骨髓来源的间充质干细胞(Human bone marrow mesenchymal stem cells,BM-h MSCs)增殖及成骨分化的影响。方法:从Scien Cell实验室购买的BM-hMSCs分别在无血清生长培养基(对照组)和添加4mmol(4P组)、8mmol(8P组)磷离子的无血清生长培养基中培养21天,通过CCK8比色法评估细胞的增殖情况;RT-PCR检测成骨分化标记性基因胶原蛋白Ⅰ、骨钙素、碱性磷酸酶的表达水平;茜素红染色检测BM-hMSCs成骨分化产生的矿化结节。结果:在培养4、7、14天时,4P和8P组中BM-hMSCs的增殖都明显高于对照组,且8P组中BM-h MSCs的增殖高于4P组;培养21天时,4P和8P组中BM-hMSCs的增殖明显低于对照组。与对照组相比,在培养7天时,4P和8P组OC的表达下调,而14、21天时,OC的表达上调。4P和8P组中ALP的表达水平与对照组无明显差异。7天时,4P、8P组ColⅠ的表达水平均明显高与对照组;14天时,8P组ColⅠ的表达水平与对照组表达无明显差异;21天时,4P、8P组中ColⅠ的表达水平都均与对照组表达无明显差异。培养21天时,4P、8P组矿化结节的形成明显增加。结论:磷离子在早期可以促进BM-hMSCs的增殖,晚期诱导其成骨分化。     

高压氧对体外培养的成骨细胞增殖和分化的影响

为探讨高压氧对成骨细胞增殖和成骨分化的影响,把来源于牙槽骨的成骨细胞接种在24孔培养皿中,每孔2 500个细胞,4个治疗组分别接受不同条件的高压氧治疗,分别是2.4ATA 90 min,2.4ATA 30 min,1.5ATA 90 min和1.5ATA30 min,每天一次,共10天.对照组进行常规的细胞培养.分别在高压氧治疗前和高压氧治疗后的1、2、3、4、6、8、10天,采用WST-1分析试剂进行成骨细胞的增殖分析.使用乳酸脱氢酶(LDH)毒性分析法检测高压氧对成骨细胞的毒性影响.另将细胞接种于96孔培养皿中,每孔10 000个细胞,正常培养3天后,改用成骨化培养基,24h后,两个治疗组分别接受2.4ATA 90 min和1.5ATA 90 min的高压氧治疗,每天一次共19次.采用钙沉积分析法、碱性磷酸酶(ALP)活性分析和Von Kossa染色进行成骨分析.同样的方法观察高压空气对细胞增殖和分化的影响.结果显示,在10%小牛血清培养基条件下,高压氧刺激了成骨细胞的增殖,而在使用2%小牛血清培养基时,并末观察到高压氧对细胞增殖的促进作用.高压氧治疗前后细胞外乳酸脱氢酶含量没有发生改变,提示了高压氧未对成骨细胞造成毒性影响.另一方面,高压氧增加了骨结节的形成,同时钙沉积增加,碱性磷酸酶的活力也显著增强,表明了高压氧促进了成骨细胞的成骨分化.     

Level of Preadipocyte Growth Factor in Rat Adipose Tissue which Specifically Permits the Proliferation of Preadipocytes Is Affected by Restricted Energy Intake

We previously reported the presence of a protein growth factor in rat adipose tissue which specifically permits the proliferation of 3T3-L1 and Obl771 preadipocytes [Biochem. Biophys. Res. Commun. 1990;171:905–912, ref. 1] and which is hereinafter referred to as PAGF (preadipocyte growth factor). In this study, the effects of long-term restricted energy intake on the PAGF activity in rat epididymal and perirenal adipose tissue toward 3T3-L1 preadipocytes were investigated. When rats were subjected to restricted energy intake for three weeks, PAGF activity increased with energy intake. The body weight, epididymal and perirenal fat depot weights and glycerol 3-phosphate dehydrogenase activity also increased with the energy intake, whereas the lactate dehydrogenase activity remained almost constant in all energy intake groups. These results suggest that the PAGF in fat depots functions in response to energy intake and contributes to the de novo formation of adipocytes and the growth of adipose tissue. This factor may provide a useful tool for further elucidation of the relationship between energy storage in adipose tissue and adipose tissue development.     

PP333对分蘖洋葱试管苗增殖和生根的影响

以MS 0.1 mg·L-1NAA 0.4 mg·L-1 6-BA为增殖培养基,1/2MS 1.5 mg·L-1IBA 0.01 mg·L-1NAA为生根培养基,添加不同浓度PP333的结果表明:增殖培养基中添加1.0 mg·L-1PP333对分蘖洋葱试管苗增殖生长有促进作用,减少超度含水态苗的发生;生根培养基中添加0.1 mg·L-1PP333对分蘖洋葱试管苗生根壮苗有良好效应.     

去乙酰化酶1基因对牛前体脂肪细胞凋亡影响的研究

去乙酰化酶1(sirtuin type1,SIRTl)是一个新的脂肪细胞调控因子,通过与其靶基因叉头转录因子1(the forkhead box O family1,FoxO1)相互作用,参与细胞增殖、分化、衰老、凋亡和代谢过程.利用吖啶橙(acridine orange,AO)染色、流式细胞仪、荧光定量PCR(quantitative real-timePCR,qPCR)等技术方法,研究SIRT1的抑制剂———烟酸胺(nicotinamide,NAM)处理后对鲁西黄牛皮下前体脂肪细胞(bovine subcutaneous preadipocytes,BSP)和肌内前体脂肪细胞(bovine intramuscular preadipocytes,BIP)凋亡的影响.观察了BSP和BIP的凋亡形态;比较了SIRT1基因抑制后,相关基因如FoxO1等在两种细胞之间的表达差异.结果表明,NAM对BSP和BIP细胞表现出相同的生长抑制作用,处理组的BSP和BIP细胞的凋亡率均显著高于对照组,其作用可能通过抑制SIRT1,激活FoxO1凋亡通路实现.SIRT1及其相关基因对BSP和BIP的调控存在不同途径.