本文選題：肌肉生長(zhǎng)抑制素　切入點(diǎn)：精氨酸　出處：《四川農(nóng)業(yè)大學(xué)》2011年博士論文　論文類型：學(xué)位論文

【摘要】：肌肉生長(zhǎng)抑制素(myostatin, MSTN)又名GDF-8,其作為骨骼肌生長(zhǎng)發(fā)育的負(fù)調(diào)控因子對(duì)動(dòng)物脂肪沉積和細(xì)胞成脂分化也具有調(diào)控作用。然而,MSTN對(duì)細(xì)胞成脂分化的調(diào)控目前仍存在不一致的報(bào)道。這可能與采用的細(xì)胞模型和研究方法不同有關(guān)。近年的研究表明,成體干細(xì)胞才是動(dòng)物組織生長(zhǎng)和修復(fù)的天然系統(tǒng),而一種干細(xì)胞不可能適于所有的研究和應(yīng)用。鑒于此,本研究開(kāi)展了以下四個(gè)試驗(yàn)以揭示MSTN在豬間充質(zhì)干細(xì)胞成脂分化中的作用,并考察MSTN在營(yíng)養(yǎng)調(diào)控豬間充質(zhì)干細(xì)胞成脂分化中的作用。 試驗(yàn)一采用流式細(xì)胞術(shù)和多向誘導(dǎo)分化等方法比較了6～7周齡健康C57BL/6J小鼠脂肪、肌肉及妊娠第16～17天胎鼠肌肉組織來(lái)源間充質(zhì)干細(xì)胞在細(xì)胞形態(tài)、免疫表型、增殖及多向分化潛能等方面的差異。結(jié)果表明,不同來(lái)源間充質(zhì)干細(xì)胞均表達(dá)間質(zhì)細(xì)胞特征標(biāo)志(CD29、CD44、CD105),但不表達(dá)造血干細(xì)胞、血細(xì)胞標(biāo)志(CD45)及內(nèi)皮祖細(xì)胞、內(nèi)皮細(xì)胞標(biāo)志(CD31、CD34)。第1代胎兒肌肉源細(xì)胞CD44陽(yáng)性率僅為27.1%,極顯著低于脂肪(61.7%)和成體肌肉源細(xì)胞(78.8%)(P0.0001),但第3代胎兒肌肉源細(xì)胞CD44表達(dá)率(99.5%)顯著增強(qiáng)。與第1代相比,第3代成體肌肉(skeletal muscle derived mesenchymal stem cells, MDSCs)或胎兒來(lái)源肌肉間充質(zhì)干細(xì)胞(fetal muscle derived mesenchymal stem cells, FMSCs) Sca-1 (stem cell antigen 1)表達(dá)增強(qiáng)且顯著高于脂肪來(lái)源的間充質(zhì)干細(xì)胞(adipose derived mesenchymal stem cells, ADSCs)。這一結(jié)果提示Sca-1表達(dá)可能與細(xì)胞成肌分化能力有關(guān)。多向分化潛能比較結(jié)果表明,MDSCs成肌誘導(dǎo)28天后骨骼肌特異性轉(zhuǎn)錄因子Myodl、myogenin、desmin、CKM和MyHC的mRNA表達(dá)水平均顯著高于FMSCs (P 0.01),而ADSCs成肌分化標(biāo)志基因mRNA表達(dá)水平最低且未檢出MyHC和骨骼肌特異性鈣粘蛋白(M-Cadherrin)表達(dá)。盡管ADSCs成脂誘導(dǎo)第14天生脂標(biāo)志基因均顯著高于MDSCs (P 0.01),但ADSCs生脂誘導(dǎo)28天后LPL和aP2的表達(dá)水平與MDSCs并無(wú)顯著差異(P0.05)。出乎意料的是,FMSCs成脂誘導(dǎo)后表現(xiàn)出最低的生脂能力。生成骨誘導(dǎo)4周后MDSCs和FMSCs OCN表達(dá)極顯著強(qiáng)于ADSCs(P0.01),而MDSCs和FMSCs間無(wú)顯著差異。FMSCs成骨誘導(dǎo)后OPN基因表達(dá)顯著強(qiáng)于MDSCs (P0.05)和ADSCs (P0.01)。由此可見(jiàn),MDSCs和FMSCs較ADSCs具有更強(qiáng)的成肌或成骨分化能力,ADSCs和MDSCs則表現(xiàn)出更強(qiáng)的生脂能力。因此,盡管不同來(lái)源間充質(zhì)干細(xì)胞具有相似的生物學(xué)特性,但重新認(rèn)識(shí)它們各自生物學(xué)特性的差異對(duì)于進(jìn)一步開(kāi)展相關(guān)研究十分重要。 鑒于有研究表明MSTN可替代經(jīng)典的生脂誘導(dǎo)劑地塞米松(DEX)誘導(dǎo)間充質(zhì)干細(xì)胞成脂分化,試驗(yàn)二采用經(jīng)典的細(xì)胞生物學(xué)方法鑒定了體外分離培養(yǎng)的豬骨髓(pBMSCs)、背部脂肪(pADSCs)、背最長(zhǎng)肌來(lái)源間充質(zhì)干細(xì)胞(pMDSCs),并重點(diǎn)考察MSTN和DEX對(duì)豬不同來(lái)源間充質(zhì)干細(xì)胞成脂分化的影響,以探討二者在間充質(zhì)干細(xì)胞成脂誘導(dǎo)分化中的作用。結(jié)果表明,豬不同來(lái)源間充質(zhì)干細(xì)胞在細(xì)胞形態(tài)、分子表型及多向分化潛能等方面具有間充質(zhì)干細(xì)胞的典型特征,但MSTN和DEX在其成脂分化過(guò)程中具有不同的作用。外源添加MSTN增強(qiáng)了pBMSCs PPARy2、LPL和aP2等生脂基因的表達(dá),但在無(wú)DEX的成脂誘導(dǎo)條件下MSTN對(duì)胞內(nèi)甘油三酯含量并無(wú)明顯影響。對(duì)于pADSCs和pMDSCs,無(wú)論成脂誘導(dǎo)體系是否含有DEX,MSTN均抑制了細(xì)胞脂肪沉積和生脂基因的表達(dá)(P0.01)。此外,細(xì)胞來(lái)源與試驗(yàn)處理間存在顯著的互作關(guān)系(P0.001),即不同組織來(lái)源間充質(zhì)干細(xì)胞成脂分化誘導(dǎo)時(shí)對(duì)外源添加MSTN和DEX反應(yīng)各不相同。這一結(jié)果提示,開(kāi)展相關(guān)研究,選擇適宜的細(xì)胞來(lái)源和研究方法尤為重要。 試驗(yàn)三以豬脂肪來(lái)源的間充質(zhì)干細(xì)胞為模型,研究外源添加Arg對(duì)間充質(zhì)干細(xì)胞成肌和成脂分化的影響,同時(shí)考察營(yíng)養(yǎng)調(diào)控間充質(zhì)干細(xì)胞定向分化過(guò)程中MSTN的表達(dá)規(guī)律。結(jié)果表明,成脂誘導(dǎo)條件下,添加Arg極顯著增加細(xì)胞內(nèi)甘油三酯生成量(P0.01),添加50μg/mL Arg組細(xì)胞甘油三酯含量最高,提高Arg添加水平反而有降低細(xì)胞甘油三酯含量的趨勢(shì)(P0.05)。添加Arg對(duì)細(xì)胞成肌分化基因(myogenin、desmin和CKM)和成脂分化基因(PPARy2、LPL和aP2)的表達(dá)均具有顯著的促進(jìn)作用,成肌誘導(dǎo)條件下,外源添加Arg極顯著抑制了細(xì)胞內(nèi)源MSTN的表達(dá)。這一結(jié)果表明,外源添加Arg可能通過(guò)抑制細(xì)胞MSTN的表達(dá)促進(jìn)豬間充質(zhì)干細(xì)胞成肌分化,但Arg對(duì)豬間充質(zhì)干細(xì)胞成脂分化的調(diào)控是否與MSTN表達(dá)有關(guān)仍需進(jìn)一步研究證實(shí)。 試驗(yàn)四以豬脂肪和肌肉來(lái)源間充質(zhì)干細(xì)胞作為皮下脂肪組織和肌內(nèi)脂肪的體外研究模型,采用外源添加Arg與MSTN活性蛋白或特異性抗體相結(jié)合的方式,重點(diǎn)考察MSTN在Arg調(diào)控間充質(zhì)干細(xì)胞成脂分化中的作用。試驗(yàn)結(jié)果表明,MSTN蛋白和抗體添加水平分別為100ng/mL和4μg/mL時(shí)對(duì)豬間充質(zhì)干細(xì)胞成脂分化的影響最大。添加MSTN蛋白極顯著抑制了細(xì)胞內(nèi)甘油三酯的生成(P0.01),而同時(shí)外源添加Arg可部分地緩解MSTN對(duì)間充質(zhì)干細(xì)胞胞內(nèi)脂質(zhì)沉積的抑制。添加MSTN蛋白極顯著抑制了脂肪來(lái)源間充質(zhì)干細(xì)胞ADD1、C/EBPa、PPARγ2和LPL的表達(dá)(P0.01),而外源添加Arg極顯著地增強(qiáng)了ADD1和aP2的表達(dá)(P0.01)；添加MSTN蛋白極顯著抑制了肌肉來(lái)源間充質(zhì)干細(xì)胞PPARγ2和aP2的表達(dá)(P0.01)并有抑制ADD1、C/EBPα表達(dá)水平的趨勢(shì),而Arg極顯著地增強(qiáng)了ADD1的表達(dá)(P0.01)。由此可見(jiàn),MSTN抑制了豬脂肪和肌肉來(lái)源間充質(zhì)干細(xì)胞成脂分化,而Arg對(duì)細(xì)胞成脂分化表現(xiàn)出相反的作用。盡管MSTN和Arg均可通過(guò)影響ADD1的表達(dá)調(diào)控豬脂肪和肌肉來(lái)源間充質(zhì)干細(xì)胞成脂分化,但二者的調(diào)控途徑并不相同。 綜上所述,不同來(lái)源間充質(zhì)干細(xì)胞在細(xì)胞表型、增殖和多向分化潛能等方面存在較大差異。MSTN對(duì)不同來(lái)源間充質(zhì)干細(xì)胞成脂分化也具有不同影響,MSTN促進(jìn)了豬骨髓來(lái)源間充質(zhì)干細(xì)胞成脂分化但抑制了豬脂肪和肌肉來(lái)源間充質(zhì)干細(xì)胞的成脂分化。。外源添加Arg增強(qiáng)了豬脂肪來(lái)源間充質(zhì)干細(xì)胞成肌和成脂分化,且Arg在成肌條件下抑制了細(xì)胞內(nèi)源MSTN的表達(dá)。盡管MSTN和Arg均可通過(guò)影響ADD1的表達(dá)調(diào)控間充質(zhì)干細(xì)胞成脂分化,但二者調(diào)控途徑并不相同。
[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.

【學(xué)位授予單位】：四川農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

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