Myostatin和Arg对猪间充质干细胞成脂分化的调控作用

发布时间：2018-03-03 15:43

本文选题：肌肉生长抑制素　切入点：精氨酸　出处：《四川农业大学》2011年博士论文　论文类型：学位论文

【摘要】：肌肉生长抑制素(myostatin, MSTN)又名GDF-8,其作为骨骼肌生长发育的负调控因子对动物脂肪沉积和细胞成脂分化也具有调控作用。然而,MSTN对细胞成脂分化的调控目前仍存在不一致的报道。这可能与采用的细胞模型和研究方法不同有关。近年的研究表明,成体干细胞才是动物组织生长和修复的天然系统,而一种干细胞不可能适于所有的研究和应用。鉴于此,本研究开展了以下四个试验以揭示MSTN在猪间充质干细胞成脂分化中的作用,并考察MSTN在营养调控猪间充质干细胞成脂分化中的作用。试验一采用流式细胞术和多向诱导分化等方法比较了6～7周龄健康C57BL/6J小鼠脂肪、肌肉及妊娠第16～17天胎鼠肌肉组织来源间充质干细胞在细胞形态、免疫表型、增殖及多向分化潜能等方面的差异。结果表明,不同来源间充质干细胞均表达间质细胞特征标志(CD29、CD44、CD105),但不表达造血干细胞、血细胞标志(CD45)及内皮祖细胞、内皮细胞标志(CD31、CD34)。第1代胎儿肌肉源细胞CD44阳性率仅为27.1%,极显著低于脂肪(61.7%)和成体肌肉源细胞(78.8%)(P0.0001),但第3代胎儿肌肉源细胞CD44表达率(99.5%)显著增强。与第1代相比,第3代成体肌肉(skeletal muscle derived mesenchymal stem cells, MDSCs)或胎儿来源肌肉间充质干细胞(fetal muscle derived mesenchymal stem cells, FMSCs) Sca-1 (stem cell antigen 1)表达增强且显著高于脂肪来源的间充质干细胞(adipose derived mesenchymal stem cells, ADSCs)。这一结果提示Sca-1表达可能与细胞成肌分化能力有关。多向分化潜能比较结果表明,MDSCs成肌诱导28天后骨骼肌特异性转录因子Myodl、myogenin、desmin、CKM和MyHC的mRNA表达水平均显著高于FMSCs (P 0.01),而ADSCs成肌分化标志基因mRNA表达水平最低且未检出MyHC和骨骼肌特异性钙粘蛋白(M-Cadherrin)表达。尽管ADSCs成脂诱导第14天生脂标志基因均显著高于MDSCs (P 0.01),但ADSCs生脂诱导28天后LPL和aP2的表达水平与MDSCs并无显著差异(P0.05)。出乎意料的是,FMSCs成脂诱导后表现出最低的生脂能力。生成骨诱导4周后MDSCs和FMSCs OCN表达极显著强于ADSCs(P0.01),而MDSCs和FMSCs间无显著差异。FMSCs成骨诱导后OPN基因表达显著强于MDSCs (P0.05)和ADSCs (P0.01)。由此可见,MDSCs和FMSCs较ADSCs具有更强的成肌或成骨分化能力,ADSCs和MDSCs则表现出更强的生脂能力。因此,尽管不同来源间充质干细胞具有相似的生物学特性,但重新认识它们各自生物学特性的差异对于进一步开展相关研究十分重要。鉴于有研究表明MSTN可替代经典的生脂诱导剂地塞米松(DEX)诱导间充质干细胞成脂分化,试验二采用经典的细胞生物学方法鉴定了体外分离培养的猪骨髓(pBMSCs)、背部脂肪(pADSCs)、背最长肌来源间充质干细胞(pMDSCs),并重点考察MSTN和DEX对猪不同来源间充质干细胞成脂分化的影响,以探讨二者在间充质干细胞成脂诱导分化中的作用。结果表明,猪不同来源间充质干细胞在细胞形态、分子表型及多向分化潜能等方面具有间充质干细胞的典型特征,但MSTN和DEX在其成脂分化过程中具有不同的作用。外源添加MSTN增强了pBMSCs PPARy2、LPL和aP2等生脂基因的表达,但在无DEX的成脂诱导条件下MSTN对胞内甘油三酯含量并无明显影响。对于pADSCs和pMDSCs,无论成脂诱导体系是否含有DEX,MSTN均抑制了细胞脂肪沉积和生脂基因的表达(P0.01)。此外,细胞来源与试验处理间存在显著的互作关系(P0.001),即不同组织来源间充质干细胞成脂分化诱导时对外源添加MSTN和DEX反应各不相同。这一结果提示,开展相关研究,选择适宜的细胞来源和研究方法尤为重要。试验三以猪脂肪来源的间充质干细胞为模型,研究外源添加Arg对间充质干细胞成肌和成脂分化的影响,同时考察营养调控间充质干细胞定向分化过程中MSTN的表达规律。结果表明,成脂诱导条件下,添加Arg极显著增加细胞内甘油三酯生成量(P0.01),添加50μg/mL Arg组细胞甘油三酯含量最高,提高Arg添加水平反而有降低细胞甘油三酯含量的趋势(P0.05)。添加Arg对细胞成肌分化基因(myogenin、desmin和CKM)和成脂分化基因(PPARy2、LPL和aP2)的表达均具有显著的促进作用,成肌诱导条件下,外源添加Arg极显著抑制了细胞内源MSTN的表达。这一结果表明,外源添加Arg可能通过抑制细胞MSTN的表达促进猪间充质干细胞成肌分化,但Arg对猪间充质干细胞成脂分化的调控是否与MSTN表达有关仍需进一步研究证实。试验四以猪脂肪和肌肉来源间充质干细胞作为皮下脂肪组织和肌内脂肪的体外研究模型,采用外源添加Arg与MSTN活性蛋白或特异性抗体相结合的方式,重点考察MSTN在Arg调控间充质干细胞成脂分化中的作用。试验结果表明,MSTN蛋白和抗体添加水平分别为100ng/mL和4μg/mL时对猪间充质干细胞成脂分化的影响最大。添加MSTN蛋白极显著抑制了细胞内甘油三酯的生成(P0.01),而同时外源添加Arg可部分地缓解MSTN对间充质干细胞胞内脂质沉积的抑制。添加MSTN蛋白极显著抑制了脂肪来源间充质干细胞ADD1、C/EBPa、PPARγ2和LPL的表达(P0.01),而外源添加Arg极显著地增强了ADD1和aP2的表达(P0.01)；添加MSTN蛋白极显著抑制了肌肉来源间充质干细胞PPARγ2和aP2的表达(P0.01)并有抑制ADD1、C/EBPα表达水平的趋势,而Arg极显著地增强了ADD1的表达(P0.01)。由此可见,MSTN抑制了猪脂肪和肌肉来源间充质干细胞成脂分化,而Arg对细胞成脂分化表现出相反的作用。尽管MSTN和Arg均可通过影响ADD1的表达调控猪脂肪和肌肉来源间充质干细胞成脂分化,但二者的调控途径并不相同。综上所述,不同来源间充质干细胞在细胞表型、增殖和多向分化潜能等方面存在较大差异。MSTN对不同来源间充质干细胞成脂分化也具有不同影响,MSTN促进了猪骨髓来源间充质干细胞成脂分化但抑制了猪脂肪和肌肉来源间充质干细胞的成脂分化。。外源添加Arg增强了猪脂肪来源间充质干细胞成肌和成脂分化,且Arg在成肌条件下抑制了细胞内源MSTN的表达。尽管MSTN和Arg均可通过影响ADD1的表达调控间充质干细胞成脂分化,但二者调控途径并不相同。
[Abstract]:Myostatin (myostatin, MSTN) also known as GDF-8, as a negative regulator of skeletal muscle growth and adipogenic differentiation also play a role in the regulation of animal fat deposition and cells. However, MSTN regulation of adipogenic differentiation of cells is still not consistent on different reports. This can and the cell model and research methods. Recent studies have shown that adult stem cells is the natural system of animal tissue growth and repair, and a kind of stem cells may not be suitable for research and application. In view of this, four experiments were conducted in this study revealed that the MSTN in pig mesenchymal stem cells differentiation into fat, and the effects of MSTN in the nutritional regulation of porcine mesenchymal stem cells into fat differentiation.
A test by flow cytometry and multi-directional differentiation method to compare 6~7 week old healthy C57BL/6J mice fat, muscle and pregnancy sixteenth ~ 17 days of fetal rat muscle tissue derived mesenchymal stem cells in cell morphology, phenotype, proliferation and multilineage differentiation potential differences. The results show that from different sources mesenchymal stem cells expressed markers of interstitial cells (CD29, CD44, CD105 features), but did not express hematopoietic stem cells, blood cell marker (CD45) and endothelial progenitor cells, endothelial cell markers (CD31, CD34). The positive rate of the first generation of CD44 cells from human fetal muscle was only 27.1%, significantly lower than fat (61.7%) and adult muscle derived cells (78.8%) (P0.0001), but the third generation of fetal muscle derived cells the expression rate of CD44 (99.5%) was significantly enhanced. Compared with the first generation, third generation of adult muscle (skeletal muscle derived mesenchymal stem cells, MDSCs) or fetal muscle mesenchymal Mesenchymal stem cells (fetal muscle derived mesenchymal stem cells FMSCs Sca-1 (stem) cell antigen 1) and significantly higher than the expression of adipose derived mesenchymal stem cells (adipose derived mesenchymal stem cells, ADSCs). These results suggest that Sca-1 expression may be of myogenic capability. The comparison results show that the potential of multi-directional differentiation MDSCs, 28 days after the induction of muscle skeletal muscle specific transcription factor Myodl, myogenin, desmin, CKM and MyHC expression levels of mRNA were significantly higher than FMSCs (P 0.01), while the ADSCs myoblast differentiation marker gene expression of mRNA and MyHC was not detected and the lowest level of skeletal muscle specific expression of E-cadherin (M-Cadherrin). Although ADSCs fourteenth natural fat induced adipogenic genes were significantly higher than that of MDSCs (P 0.01), but the ADSCs expression level of fat induced 28 days after LPL and aP2 and MDSCs had no significant difference (P0.05). It is, FMSCs after adipogenic induction showed adipogenic ability. The lowest bone formation after 4 weeks of induction of MDSCs and FMSCs OCN expression was significantly stronger than that of ADSCs (P0.01), while MDSCs and FMSCs showed no significant difference.FMSCs after osteogenic induction of OPN gene expression was significantly stronger than that of MDSCs (P0.05) and ADSCs (P0.01) by this. Obviously, MDSCs and FMSCs compared with ADSCs muscle or osteogenic differentiation ability stronger, ADSCs and MDSCs showed a fat more. Therefore, despite the different sources of mesenchymal stem cells have similar biological characteristics and differences to understand their unique biological characteristics is very important for the further research.
In view of studies have shown that adipogenic inducer dexamethasone can replace the classical MSTN (DEX) induced mesenchymal stem cell adipogenic differentiation, cell biology test two using the classical methods of in vitro cultured porcine bone marrow identification (pBMSCs), back fat (pADSCs), longissimus muscle derived mesenchymal stem cells (pMDSCs), and focuses on the MSTN and DEX on different sources of porcine mesenchymal stem cells adipogenic differentiation, to explore the two in mesenchymal stem cells adipogenic differentiation in vitro. The results show that the pigs of different sources of mesenchymal stem cells in cell morphology, molecular phenotype and multilineage differentiation potential has typical characteristics of mesenchymal stem cells, but MSTN and DEX have different role in adipogenic differentiation. Exogenous MSTN enhanced the expression of LPL and pBMSCs PPARy2, aP2 and other lipogenic genes, but no DEX in adipogenic induced MSTN on intracellular conditions Triglyceride content has no obvious effect. For pADSCs and pMDSCs, regardless of whether fat induced system containing DEX, the expression of MSTN was inhibited by cell fat deposition and lipogenetic genes (P0.01). In addition, there was significant interaction between cells and experimental treatments (P0.001), different tissue derived mesenchymal stem cells adipogenic induction of exogenous MSTN and DEX reaction is different. This result suggests that, to carry out related research, selection of suitable cell sources and research methods is particularly important.
Test of three porcine adipose derived mesenchymal stem cells as a model of exogenous Arg on muscle and adipogenic differentiation of mesenchymal stem cells, and the effects of nutritional regulation of mesenchymal stem cell differentiation and expression of MSTN in the process. The results show that the adipogenic conditions, adding Arg increased production of intracellular triglyceride (P0.01), adding 50 g/mL cells in Arg group triglyceride content was the highest, improve the level of dietary Arg but decreased cell triglyceride content trend (P0.05). Add Arg on cell myogenic differentiation genes (myogenin, desmin and CKM) and adipogenic differentiation genes (PPARy2, LPL and aP2) expression has a significant role in promoting myogenic differentiation conditions, exogenous Arg significantly inhibited the expression of endogenous MSTN. The results showed that exogenous Arg could promote pig mesenchymal cells by inhibiting expression of MSTN Stem cells have myogenic differentiation, but it is still necessary to further study whether the regulation of lipid differentiation of porcine mesenchymal stem cells (Arg) is related to MSTN expression.
Experiment four with pig fat and muscle derived mesenchymal stem cells as in vitro model of subcutaneous adipose tissue and intramuscular fat, with exogenous Arg and MSTN activity or protein specific antibody combination, focusing on MSTN Arg in the regulation of mesenchymal stem cells into fat differentiation. Experimental results show that MSTN, protein and antibody levels were 100ng/mL and 4 g/mL of porcine mesenchymal stem cells adipogenic differentiation. Adding MSTN protein significantly inhibited the formation of intracellular triglyceride (P0.01), while exogenous Arg could partially alleviate MSTN on mesenchymal stem inhibiting lipid deposition intracellular. Add MSTN protein significantly restrained adipose derived mesenchymal stem cells ADD1, C/EBPa, expression of PPAR gamma 2 and LPL (P0.01), while exogenous Arg significantly enhanced the expression of ADD1 and aP2 (P0.01); add MSTN egg White significant inhibition of muscle derived mesenchymal stem cells PPAR expression 2 and aP2 (P0.01) and the inhibition of ADD1, C/EBP expression level of the trend, while Arg significantly enhanced the expression of ADD1 (P0.01). Thus, MSTN inhibited the pig fat and muscle derived mesenchymal stem cells adipogenic differentiation, Arg cells to adipogenic differentiation showed the opposite effect. Although MSTN and Arg can influence the ADD1 expression regulation of pig fat and muscle derived mesenchymal stem cell adipogenic differentiation, but regulatory pathways of the two are not the same.
In conclusion, different sources of mesenchymal stem cells in cell phenotype, there is a big difference in.MSTN also has different effects on different sources of mesenchymal stem cells into adipocytes proliferation and differentiation potential, MSTN promotes the pig bone marrow derived mesenchymal stem cells but inhibit the adipogenic differentiation of porcine adipose and muscle sources mesenchymal stem cells to adipogenic differentiation. Exogenous Arg enhances the porcine adipose derived mesenchymal stem cells into muscle and adipose differentiation, and Arg in myogenic conditions inhibited the expression of endogenous MSTN. Although MSTN and Arg are affected by expression of ADD1 controlled mesenchymal stem cell adipogenic differentiation however, the two regulation is not the same way.

【学位授予单位】：四川农业大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R329

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