【作者】 刘涛；

【导师】 马秋刚；

【作者基本信息】 中国农业大学 ， 动物营养与饲料科学， 2017， 博士

【摘要】 骨骼肌卫星细胞在肌肉生长发育、再生和损伤修复中扮演着非常重要的角色。本试验主要研究L-肌肽对骨骼肌卫星细胞增殖和分化的影响,揭示L-肌肽促进猪骨骼肌卫星细胞增值和分化的机理。试验一选取健康1日龄的长白猪,优化分离程序,分离猪背最长肌卫星细胞,采用差速贴壁法对分离得到的卫星细胞进行纯化;接种96孔板,培养24 h后,用含0、0.2、2、1、10、20和40 mM/L的L-肌肽的细胞培养基进行处理,每个处理10个重复。结果显示:与对照组相比,0.2、1和2 mM/L的L-肌肽显著提高了(P<0.05)卫星细胞接种72、96和120 h的OD值,40 mM/L的L-肌肽显著抑制了(P<0.05)卫星细胞接种120 h的OD值,对于其他时间点的OD值虽然具有抑制作用,但差异不显著(P>0.05)。这说明0.2、1和2 mM/L促进了卫星细胞增值,而40 mM/L的L-肌肽具有抑制卫星细胞的增值的作用。因此,选择0、0.2、2和20 mM/L的L-肌肽进行后续试验。试验二取生长良好的第3代卫星细胞,接种96/6孔板,培养24 h后,分别用含0、0.2、2和20 mM/L的L-肌肽、β-丙氨酸、L-组氨酸以及两种氨基酸混合物的细胞培养基进行处理,每个处理10个重复,研究L-肌肽及其组分对猪背最长肌卫星细胞增殖的影响。结果表明.:与对照组相比,0.2和2 mM/L的L-肌肽显著提高了(P<0.05)卫星细胞接种72、96和120 h的OD值,促进了卫星细胞的增殖,而单独或混合添加0.2和2 mM/L的β-丙氨酸和L-组氨酸对卫星细胞的增殖没有显著影响(P>0.05);20 mM/L的L-肌肽和β-丙氨酸对卫星细胞增殖没有显著影响(P>0.05),而20 mM/L的L-组氨酸显著抑制了(P<0.05)卫星细胞增殖;0.2和2 mM/L的L-肌肽显著降低了(P<0.05)卫星细胞接种96 h时G0/G1期细胞比例及MyoD基因mRNA的相对表达量,提高S期细胞比例及IGF-1基因mRNA的相对表达量(P<0.05)。各处理浓度对卫星细胞的大小没有显著影响(P>0.05)。试验三取生长良好的第3代卫星细胞,接种6孔板,培养24 h后,分别用含0和2 mM/L的L-肌肽的细胞培养基处理,每个处理4个重复。在接种的96 h,提取蛋白,研究L-肌肽对猪背最长肌卫星细胞增殖期m TOR信号通路的影响。结果显示:L-肌肽显著提高了增殖期IGF-1、Akt、mTOR、S6K蛋白的表达(P<0.05),激活了 mTOR及其下游S6K,促进了蛋白质的合成。试验四取生长良好的第3代卫星细胞,接种6孔板,当细胞铺满板的80%左右时,换成含不同浓度L-肌肽的的分化培养基,测定其对卫星细胞分化融合率、迁移能力、MyHC的表达及Wnt/β-catenin信号通路的影响。结果表明:0.2和2mM/L的L-肌肽可显著提高(P<0.05)卫星细胞分化24、48、72、96和120h的融合率及迁移能力;Western试验结果显示L-肌肽提高了分化96h时MyHC的表达(P<0.05),激活了 Wnt/β-catenin信号通路。综上,L-肌肽促进卫星细胞增殖的机理是其激活了 m TOR信号通路,加速了蛋白质的合成,提高了 S期细胞比例;L-肌肽促进卫星细胞分化的机理是通过激活Wnt/β-catenin信号通路实现的。更多还原

【Abstract】 Satellite cells(SCs)play an important role in post-natal muscle growth and regeneration,the purpose of this study was conducted to investigate the effects of L-carnosine on proliferation and differentiation of SCs,revealing the underlying mechanism.Experiment 1:One-day old and healthy landrace pigs were selected to isolated SCs in this study,and then the SCs were purified via differential adhesion method;SCs were seeded in 96-well plates at a density of 1×104 cells/mL in DMEM/F12 medium with 10%FBS(GM)).24 h after seeding,the cultures were treated with fresh GM containing different levels of L-carnosine.The MTT assay showed that supplementation of L-carnosine at dose of 0.2,2,1 and 2 mM/L significantly increased(P<0.05)proliferation,which was significantly inhibited(P<0.05)by 20 mM/L of L-carnosine.Therefore,0,0.2,2 and 20 mM/L were selected for the nest experiments.Experiment 2:SCs at the third generation were seeded in 96/6-well plates.24 h after seeding,the cultures were treated with fresh GM containing different levels of L-carnosine(0,0.2,2 and 20 mM/L).The effects of carnosine on cell proliferation were determined at 24,48,72,96 and 120 h after seeding.The results showed that:supplementation of L-carnosine at dose of 0.2 and 2 mM/L significantly increased(P<0.05)the SCs proliferation at 72,96 and 120 h after seeding,but single or mixed addition of β-alanine and L-histidine did not significantly(P>0.05)affect SCs proliferation as compared to the control;SCs proliferation,which were significantly decreased by 20 mM/L of L-histidine(P<0.05),were not affected(P>0.05)by 20 mM/L of L-carnosine or β-alanine.In addition,supplementation of L-carnosine at 2 mM/L significantly decreased(P<0.05)the rate of SCs in G0/G1 phase and the relative expression of Myod gene,while significantly increased(P<0.05)the rate of SCs in S phase and the relative expression of IGF-1 gene.SCs size was not significantly affected(P>0.05)by L-carnosine.Experiment 3:SCs at the third generation were cultured in 96-well plates.24 h after seeding,the cultures were treated with fresh GM containing different levels of L-carnosine(0,and 2 mM/L).The effects of carnosine on m TOR signaling pathway were determined by Western Blot at 96 h after seeding.The results showed that the expressed levels of IGF-1、Akt、m TOR、S6K were significantly increased(P<0.05)by L-carnosine compared with the control.Experiment 4:SCs at the third generation were cultured in 96-well plates.24 h after seeding,the cultures were treated with fresh differentiation medium containing different levels of L-carnosine(0,0.2,2 and 20 mM/L).The effects of carnosine on cell differetiation were investigated.The results showed that supplementation of L-carnosine at 0.2 and 2 mM/L significantly improved the fusion rates of SCs at 24,48,72,96 and 120 h after inducing,as well as the ability of migration of SCs.Western Blot assay indicated that L-carnosine(2 mM/L)significantly increased(P<0.05)the expression of fast-,slow-and total-MyHC,and the Wnt/β-catenin signaling pathway was activated by L-carnosine(P<0.05).更多还原