Myogenic and adipogenic properties of goat skeletal muscle stem cells

The aims of the present study were to establish a culture system for goat skeletal muscle stem cells and to examine their myogenic and adipogenic properties in vitro. Cells were isolated from the skeletal muscle of the Shiba goat and cultured in vitro. Most of the cells were positive for myogenic markers, such as Pax7, MyoD, and desmin, and immunocytochemistry revealed they differentiated to form myotubes expressing myosin heavy chain, indicating they were highly myogenic. Myogenic differentiation was strongly suppressed by the addition of basic fibroblast growth factor, while proliferation was unaffected. When the cells were cultured in adipogenic differentiation medium, some of the cells differentiated into mature adipocytes that stained with Oil Red-O. These cells were immunocytochemically positive for adipogenic markers, including peroxisome proliferator-activated receptor-gamma (PPAR gamma) and CCAAT/enhancer-binding protein-alpha (C/EBP alpha). These results clearly demonstrate the presence of both myogenic and adipogenic stem cells in goat skeletal muscle.     

添加环格列酮对延边黄牛骨骼肌卫星细胞成脂转分化的影响

试验旨在探究过氧化物酶体增殖物激活受体（PPARγ）激动剂（环格列酮）对延边黄牛骨骼肌卫星细胞成脂转分化的影响。试验分为4组：对照组（CON，不添加环格列酮）、CL组（5 μmol/L环格列酮）、CM组（10 μmol/L环格列酮）和CH组（20 μmol/L环格列酮），利用不同浓度环格列酮处理骨骼肌卫星细胞96 h后，通过油红O染色法和甘油三酯的测定来验证脂滴的形成，并使用实时荧光定量PCR和Western blotting测定基因表达和蛋白质定量的变化来验证脂肪细胞的生成。甘油三酯测定和油红O染色显示，与对照组相比，添加环格列酮后，骨骼肌卫星细胞内有脂滴生成，且脂滴形成量和甘油积累量与环格列酮添加量呈正相关。实时荧光定量PCR和Western blotting结果表明，与对照组相比，环格列酮组显著增加了成脂转录因子PPARγ、CCAAT/增强子结合蛋白α（C/EBPα）和围脂滴蛋白-2（PLIN2）基因的表达，显著下调了成肌相关因子MyoD的表达（P<0.05），且所有蛋白与基因表达趋势保持一致，表明环格列酮可以促进牛骨骼肌卫星细胞向脂肪细胞分化。     

Differentiation of bovine intramuscular and subcutaneous stromal-vascular cells exposed to dexamethasone and troglitazone

The objectives of these experiments were to compare differentiation of bovine stromal-vascular (S-V) cells isolated from i.m. and s.c. adipose tissues in response to a glucocorticoid and a peroxisome proliferator-activated receptor gamma agonist. Stromal-vascular cells were isolated from i.m. and s.c. fat depots of 3 Angus steers and propagated in culture. Cells were exposed to differentiation media containing 0.25 microM dexamethasone (DEX), a glucocorticoid analog, and 40 microM troglitazone (TRO), a peroxisome proliferator-activated receptor gamma agonist, or both. Cells treated with DEX and TRO had greater (P < 0.02) glycerol-3-phosphate dehydrogenase activity than control cells. No interactions between DEX, TRO, and depot (P > 0.59) or depot differences (P = 0.41) in glycerol-3-phosphate dehydrogenase activity were found. Morphological assessment of adipogenic colonies showed that DEX induced a 1.8-fold increase in the percentage of adipogenic colonies (P = 0.03), whereas TRO increased the proportion of adipogenic colonies by 1.9-fold (P = 0.02) compared with those not treated with DEX or TRO, respectively. Depots had a similar percentage of adipogenic colonies (P = 0.18); however, the percentage of differentiated cells within adipogenic colonies was found to be 6.4-fold greater in s.c. isolates compared with i.m. (P < 0.001). Addition of TRO increased the proportion of differentiated cells within colonies by 10-fold compared with those of nontreated colonies (P < 0.001), whereas the percentage of differentiated cells within adipogenic colonies only tended to be increased by DEX (P = 0.10). These data indicate that bovine i.m. and s.c. S-V cells are capable of enhanced differentiation in response to DEX and TRO, and these effects were additive. Most importantly, inherent differences in the capacity to differentiate exist between adipogenic bovine i.m. and s.c. S-V cells.     

不同浓度油酸对延边牛骨骼肌卫星细胞成脂转分化的影响

试验旨在探究油酸对延边牛骨骼肌卫星细胞成脂转分化的影响。试验设1个空白对照组（CON）和3个油酸（OA）诱导组：50、100、200 μmol/L油酸组（OAL、OAM、OAH）。油酸诱导96 h后,通过测定细胞大小及活力评估油酸对细胞的影响。通过油红O染色和测定甘油三酯来验证脂滴的形成,通过实时荧光定量PCR测定相关成肌成脂基因的表达水平来验证脂肪细胞的生成。结果显示,与对照组相比,添加油酸后,延边牛骨骼肌卫星细胞内有脂滴生成,并且脂滴形成量、甘油三酯累积量与油酸呈剂量依赖关系;实时荧光定量PCR测定结果表明,成肌相关因子Pax3、MyoD显著下调（P<0.05）,成脂相关因子C/EBPβ、PPARγ显著上调（P<0.05）,脂肪酸代谢相关因子SCD显著下调（P<0.05）,PLIN2基因显著上调（P<0.05）。综合上述试验结果,用油酸诱导处理延边牛骨骼肌卫星细胞可促进细胞的成脂转分化。     

miR-186-5p调控猪原代前体脂肪细胞增殖和分化的研究

旨在探究miR-186-5p对猪原代前体脂肪细胞增殖和成脂分化的调控作用及机制.本研究采集7日龄健康马身猪公猪的颈部皮下脂肪,分离培养马身猪原代前体脂肪细胞;猪原代前体脂肪细胞分为4组,分别转染miR-186-5p模拟物(mimics)及其对照组(mimics NC),miR-186-5p抑制剂(inhibitor)及...     

Optimization of adipogenic differentiation conditions for canine adipose-derived stem cells

BackgroundCanine adipose-derived stem cells (cADSCs) exhibit various differentiation properties and are isolated from the canine subcutaneous fat. Although cADSCs are valuable as tools for research on adipogenic differentiation, studies focusing on adipogenic differentiation methods and the underlying mechanisms are still lacking.ObjectivesIn this study, we aimed to establish an optimal method for adipogenic differentiation conditions of cADSCs and evaluate the role of peroxisome proliferator-activated receptor gamma (PPARγ) and estrogen receptor (ER) signaling in the adipogenic differentiation.MethodsTo induce adipogenic differentiation of cADSCs, 3 different adipogenic medium conditions, MDI, DRI, and MDRI, using 3-isobutyl-1-methylxanthine (M), dexamethasone (D), insulin (I), and rosiglitazone (R) were tested.ResultsMDRI, addition of PPARγ agonist rosiglitazone to MDI, was the most significantly facilitated cADSC into adipocyte. GW9662, an antagonist of PPARγ, significantly reduced adipogenic differentiation induced by rosiglitazone. Adipogenic differentiation was also stimulated when 17β-estradiol was added to MDI and DRI, and this stimulation was inhibited by the ER antagonist ICI182,780.ConclusionsTaken together, our results suggest that PPARγ and ER signaling are related to the adipogenic differentiation of cADSCs. This study could provide basic information for future research on obesity or anti-obesity mechanisms in dogs.     

不同种类脂肪酸对延边黄牛骨骼肌卫星细胞成脂转分化的影响

试验旨在探究用同一浓度不同种类的脂肪酸单体（油酸、硬脂酸、棕榈油酸及棕榈酸）处理延边黄牛骨骼肌卫星细胞（BSC）,研究其对脂肪生成相关基因表达及脂滴形成的影响。从18月龄延边黄牛半膜肌中分离提取骨骼肌卫星细胞进行体外培养,在分化培养基中分别添加100 μmol/L油酸（OA）、硬脂酸（SA）、棕榈油酸（POA）和棕榈酸（PA）培养96 h,油红O染色观察脂滴生成情况,并利用实时荧光定量PCR法检测与脂肪生成相关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1（SREBP1）、硬脂酰辅酶A去饱和酶（SCD）、CCAAT/增强子结合蛋白α（C/EBPα）的表达。油红O染色结果显示,与对照组相比,所有的脂肪酸处理组细胞均有脂滴形成,油酸和棕榈油酸处理组相对于棕榈酸和硬脂酸处理组在肌管内形成的脂滴数量更多,且脂滴的形态较大。实时荧光定量PCR结果显示,在延边黄牛骨骼肌卫星细胞中添加油酸和棕榈油酸等不饱和脂肪酸增加了与脂肪合成相关基因PPARγ、SREBP1、C/EBPα的表达,抑制了SCD基因的表达;添加饱和脂肪酸（硬脂酸和棕榈酸）则在促进PPARγ、SREBP1、C/EBPα基因表达的同时也显著增加了SCD基因的表达（P < 0.05）。结果表明,添加脂肪酸可以诱导延边黄牛骨骼肌卫星细胞向脂肪细胞转分化。     

LiCl激活经典Wnt信号通路诱导猪骨骼肌卫星细胞向慢肌分化

Wnt/β-catenin信号通路参与调控细胞的增殖及分化,决定细胞的命运。LiCl通过抑制GSK-3β(glycogen synthetase kinase-3β)稳定细胞内β-catenin的表达。为研究Wnt/β-catenin信号通路在哺乳动物骨骼肌卫星细胞成肌分化过程中的作用。以原代培养猪骨骼肌卫星细胞为实验材料,将其培养在含有1%CEE和20%胎牛血清的GMEM培养液中,再用15 mmol/L LiCl诱导分化,并采用免疫荧光染色和Western blotting等方法检测经典Wnt信号通路中相关因子及成肌分化过程中细胞的蛋白表达。结果表明:LiCl处理猪骨骼肌卫星细胞后促进分化,p-GSK-3β、β-catenin、myosin及慢肌蛋白增加,p-β-catenin、GSK-3β及快肌蛋白减少,并且核内源性β-catenin增多。结果提示,激活Wnt/β-catenin信号通路促进骨骼肌卫星细胞成肌分化,并且诱导其向慢肌分化。     

盘羊脐带间充质干细胞的分离培养及诱导分化

试验旨在研究野生盘羊脐带间充质干细胞（umbilical cord mesenchymal stem cells,UC-MSCs）的分离、培养及体外多向分化潜能等生物学特性。取盘羊分娩后的新生雄性胎儿脐带,采用组织块培养法分离盘羊UC-MSCs后进行体外培养,并对UC-MSCs进行成骨、成软骨和成脂诱导分化,检测其多向分化潜能。结果显示,应用组织块培养法获得的盘羊UC-MSCs具有UC-MSCs特有的呈簇、成纤维状的特性,细胞呈"S"型曲线生长,细胞的倍增时间平均为33.50 h,且可以向成骨细胞、成软骨细胞和成脂细胞分化。诱导分化特异性染色结果表明,野生盘羊UC-MSCs经诱导后的成骨细胞经茜素红S染色呈现典型的橙红色钙沉积物聚集,成软骨细胞经阿利新蓝染色呈现蓝色的软骨基质,成脂细胞经油红O染色呈现深红色的脂滴。实时荧光定量PCR检测结果发现,随着诱导时间的延长,成骨分化的细胞中骨内γ-羧基谷氨酸蛋白（BGLAP）、骨桥蛋白基因（OPN）、Runt相关转录因子2（RUNX2）基因的相对表达量极显著升高（P<0.01）;成软骨分化的细胞中光蛋白聚糖（LUM）基因的表达量明显增加,而双糖链蛋白多糖（BGN）和Y染色体性别决定区基因盒9（SOX9）基因表达量极显著提高（P<0.01）;成脂分化的细胞中脂蛋白酶（LPL）和过氧化物酶体增殖因子活化受体γ（PPAR-γ）基因的相对表达量极显著上升（P<0.01）。试验结果表明,用盘羊胎盘附带的脐带组织可以分离到盘羊UC-MSCs,且分离到的UC-MSCs具有分化为成骨细胞、成软骨细胞及成脂细胞等多向分化潜能。     

Effect of diacylglycerol acyltransferase 2 overexpression in 3T3-L1 is associated to an increase in mono-unsaturated fatty acid accumulation

Background

Fatty acid (FA) composition is the most important parameter affecting the flavor and nutritional value of the meat. The final and the only committed step in the biosynthesis of triglycerides is catalyzed by diacylglycerol acyltransferase 2 (DGAT2). The role of DGAT2 in lipid accumulation has been demonstrated in adipocytes, However, little is known about the effect of DGAT2 on the FA composition of these cells.

Methods

To investigate the role of DGAT2 in regulating lipid accumulation, FA composition and the expression of adipogenic genes, we cloned the open reading frame of the porcine DGAT2 gene and established 3T3-L1 cells that overexpressed DGAT2. Cells were then cultured in differentiation medium (DM) without FA, with a mixture of FAs (FA-DM), or containing a ¹³C stable isotope-labeled FA mixture (IFA-DM). The FA composition of adipocytes was analyzed by gas chromatography–mass spectrometry and gas chromatography-isotope ratio mass spectrometry. Quantitative PCR and western blotting were employed to detect expression of adipogenic genes in 3T3-L1 adipocytes cultured with FA-DM for 12 d.

Results

The triacylglyceride (TAG) content was significantly higher in 3T3-L1 adipocytes overexpressing DGAT2 than in control cells. When cultured in DM or FA-DM for 12 d, cells overexpressing DGAT2 showed a higher proportion of unsaturated FAs (C16:1 and C18:1). However, when cells overexpressing DGAT2 were cultured with FA-DM for 30 min, the FA composition was almost identical to that of controls. Further, the proportion of stable isotope-labeled FAs were similar in 3T3-L1 adipocytes overexpressing DGAT2 and control cells cultured in IFA-DM for 12 d. These results collectively indicate that the higher proportion of mono-unsaturated FAs, C16:1 and C18:1, may originate from de novo FA synthesis but not from the uptake of specific FAs from the medium. This hypothesis is further supported by evidence that both mRNA and protein expression of genes involved in FA synthesis (ACACA, FASN, SCD1, and A-FABP) were significantly higher in cells overexpressing DGAT2 than in control cells.

Conclusions

In conclusion, our study revealed that TAG accumulation, the proportion of MUFAs, and the expression of adipogenic genes were higher in 3T3-L1 cells overexpressing DGAT2 than in control cells.     

Adipogenic potential of satellite cells from distinct skeletal muscle origins in the rat

The possible relationship between myofiber type composition and adipose tissue development in skeletal muscle in vivo has been suggested. Recent evidence indicated that satellite cells are multipotent cells that can undergo not only myogenic, but also adipogenic differentiation. In the present study, rat satellite cells were isolated from soleus, back, extensor digitorum longus, tibialis anterior and quadriceps muscles, and their adipogenic potentials were compared by culturing them under adipogenic conditions in vitro. Cells from soleus muscle exhibited the highest adipogenic potential as judged from Oil Red-staining and immunocytochemical C/EBPalpha-staining. The adipogenic potential of satellite cells was positively correlated with type I myofiber distribution in the corresponding muscle of origin. These results demonstrated that the adipogenic potential of satellite cells differs according to the muscle of origin and suggested that its possible correlation to type I myofiber distribution may account for preferential adipose tissue development in slow oxidative muscles.     

葛根素对Akt通路调控3T3-L1脂肪细胞分化的影响

为了研究葛根素（puerarin）对3T3-L1前体脂肪细胞分化的影响,探索其在成脂分化过程中的潜在作用机制,试验在脂肪形成过程中将0、10、50 μmol/L葛根素加入到诱导分化培养基中诱导分化,分别通过油红O染色法及甘油三酯酶法检测葛根素对3T3-L1脂肪细胞的脂滴积累、甘油三酯的影响;采用实时荧光定量PCR检测脂肪细胞中CCAAT-增强子结合蛋白α（C/EBPα）和过氧化物酶体增殖物激活受体γ（PPARγ）的mRNA表达量,Western blotting检测脂肪形成相关转录因子及Akt信号通路的蛋白水平的表达量。结果表明,10 μmol/L葛根素极显著增加了成熟脂肪细胞中脂滴和甘油三酯（TG）的积聚,极显著促进了脂肪形成相关转录因子C/EBPα和PPARγ的mRNA和蛋白水平的表达量（P<0.01）。进一步研究发现,与对照组相比,葛根素的刺激可增强Akt信号通路Ser473蛋白的磷酸化表达水平,表明葛根素对成脂分化过程的促进作用很大程度上是通过Akt信号通路的磷酸化来实现的。综上所述,葛根素能够促进3T3-L1前体脂肪细胞的分化,改善胰岛素敏感性,其作用机制与激活Akt信号通路Ser473位点的磷酸化水平有关。本试验结果可为研究胰岛素的效应机制提供新见解,为胰岛素抵抗相关疾病的治疗提供新思路。     

葛根素对松辽黑猪前体脂肪细胞成脂分化的影响

为探究葛根素对松辽黑猪前体脂肪细胞成脂分化的调控作用,在细胞诱导液中分别添加0、10、20、40、60和80 μmol/L葛根素进行成脂诱导分化,用油红O染色法和甘油三酯酶法检测脂肪细胞分化过程中脂滴聚集情况和甘油三酯含量以考察脂质沉积和分化效果,并确定葛根素的最佳添加浓度;用实时荧光定量PCR检测对照组(0 μmol/L)和最佳葛根素浓度添加组成脂标志基因细胞过氧化物酶体增殖物激活受体γ(PPARγ)、CCAAT-增强子结合蛋白α(C/EBPα)及成脂分化基因乙酰辅酶A羧化酶(ACC)、脂肪酸结合蛋白(FABP4)、应激蛋白(TRIB)和叉头框蛋白O1 (FOXO1)的mRNA的表达水平,用Western blotting检测PPARγ和C/EBPα的蛋白表达水平。结果表明,与对照组相比,20、40和60 μmol/L葛根素均显著增加脂滴和甘油三酯含量(P<0.05),且40 μmol/L葛根素效果最佳;实时荧光定量PCR结果表明,与对照组相比,40 μmol/L葛根素显著上调成脂标志基因PPARγ、C/EBPα及成脂分化基因ACC、FABP4、FOXO1和TRIB的表达(P<0.05);Western blotting结果显示,与对照组相比,40 μmol/L葛根素显著增加PPARγ蛋白表达(P<0.05)。综上所述,40 μmol/L葛根素能够促进松辽黑猪前体脂肪细胞的成脂分化和脂质沉积。     

Further insights into the characterization of equine adipose tissue-derived mesenchymal stem cells

Adipose tissue-derived stem cells (ADSCs) represent a promising subpopulation of adult stem cells for tissue engineering applications in veterinary medicine. In this study we focused on the morphological and molecular biological properties of the ADSCs. The expression of stem cell markers Oct4, Nanog and the surface markers CD90 and CD105 were detected using RT-PCR. ADSCs showed a proliferative potential and were capable of adipogenic and osteogenic differentiation. Expression of Alkaline phosphatase (AP), phosphoprotein (SPP1), Runx2 and osteocalcin (OC) mRNA were positive in osteogenic lineages and peroxisome proliferator activated receptor (Pparγ2) mRNA was positive in adipogenic lineages. ADSCs show stem cell and surface marker profiles and differentiation characteristics that are similar to but distinct from other adult stem cells, such as bone marrow-derived mesenchymal stem cells (BM-MSCs). The availability of an easily accessible and reproducible cell source may greatly facilitate the development of stem cell based tissue engineering and therapies for regenerative equine medicine.