Wnt/β-catenin信号通路对猪肌卫星细胞增殖分化影响的初步研究
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摘要
骨骼肌组织中的成肌细胞是以肌卫星细胞的形式而存在,是肌肉组织的前体细胞,在骨骼肌的生长、发育、损伤及移植中具有重要作用。研究如何有效控制肌卫星细胞的分化,使骨骼肌朝着良性发展具有很重要的实践意义。Wnt蛋白是一种由Wnt基因编码的分泌型蛋白,通过自分泌或旁分泌作用与位于细胞膜上的受体相结合,激活细胞内信号通路,调节靶基因的表达,调控胚胎的早期发育,而且与细胞极性建立、细胞命运决定等多个发育事件有关。此外,它在高等动物的胚胎发育过程中主要参与细胞的增殖、分化、极化、凋亡与抗凋亡等。Wnt信号在胚胎发生中肌肉形成过程中是必要的,是肌细胞终末分化和卫星细胞定向的关键调控因子。Wnt10b能特异性的调控肌细胞和再生肌肉的生脂潜能,并且激活Wnt10b可以抑制脂肪细胞分化。
本试验以初生健康长白仔猪为试验动物,采用RT-PCR技术成功克隆猪Wnt10b基因与Wnt/β-catenin信号通路关键因子TCF4;分离培养骨骼肌肌卫星细胞,Wnt/β-catenin信号通路激活剂25mM LiCl处理,采用四甲基偶氮唑蓝(MTT)比色、HE染色、半定量RT-PCR和Western Blot等方法,研究Wnt/β-catenin信号通路对肌卫星细胞增殖分化的影响,并对其机理进行初步探讨。主要获得以下研究结果:
1.克隆了猪Wnt10b基因部分CDS序列1190bp,包含起始密码子ATG。向GenBank提交,获得登录号EU181371,并对其进行生物信息学分析,发现与GenBank上已公布的人、小鼠、大鼠、牛、马和黑猩猩核苷酸的同源性分别为94%、89%、89%、92%、93%和92%;氨基酸同源性分别为98%、96%、96%、94%、98%和97%。表明该基因在不同物种间具有很高的保守性。该段基因编码394个氨基酸,具有Wnt家族保守的WNT1功能结构域和糖基化位点。
2. RT-PCR检测Wnt10b在猪不同组织中的表达差异,结果表明该基因在猪肝脏中表达量最高,心脏、肌肉组织、皮下脂肪以及脾脏中也表达。
3.成功克隆Wnt/β-catenin通路关键因子TCF4基因全长CDS序列2015bp,提交GenBank获得登录号EU694100。与GenBank上已公布的人、小鼠、大鼠、猕猴、牛和马的TCF4基因进行比对,同源性依次为94 %、90%、89%、93%、93%和95%。生物信息学分析发现开放式阅读框位于1-1281位。
4. 25mM Wnt/β-catenin信号通路激活剂LiCl处理猪骨胳肌细胞,发现能促进骨骼肌细胞的成肌分化,而且发现随着成肌分化的进行,β-catenin蛋白的表达量逐渐上升。
In skeletal muscle tissues, myoblasts which are precursor cells of muscle tissue exist in the form of muscle satellite cells, and plays an important role in the skeletal muscle growth, development, injury and transplantation. It is practically significant to research on how to control the differentiation of muscle satellite cell effectively. Wnt protein is a kind of secreting protein that were encoded by the Wnt genes. After binding with its receptors located on the cell membrane through an auto/paracrine manner, wnt protein activate downstream cell signaling pathways,regulate the expression of target gene and early embryonic development, cell polarity and cell fate decision. In addition, Wnt signaling pathway is mainly involved in the process of cell proliferation, differentiation, polarization, apoptosis and anti-apoptosis during the embryonic development. What’s more, Wnt signaling pathway is necessary for the muscle formation process during the embryonic development, and the key regulatory factor of myoblast terminal differentiation and muscle satellite cell decision. Wnt10b can pecifically regulate adipogenesis potential of myogenesis and regenerated muscle, and activation of Wnt10b can inhibit adipocyte differentiation.
In this study, primary health Landrace pigs were adopted as experimental animals. Then Wnt10b gene and TCF4 which was key factor of Wnt/β-catenin signaling pathway were successfully cloned using RT-PCR technique. Skeletal muscle satellite cells were isolated, and treated with LiCl (25 mM) which was activator of Wnt/β-catenin signaling pathway, then a our-methyl-tetrazolium (MTT) colorimetric method, HE staining, HE staining, semi-quantitative RT-PCR and Western Blot methods was applied to study the effects of Wnt/β-catenin signaling pathway on muscle satellite cell proliferation and differentiation ,and discussed their mechanism. The following were the major findings:
1. 1190 bp of partially pig Wnt10b gene CDS sequence was Cloned, including start codon ATG. Submitted to GenBank, gain a login number EU181371. After an bioinformatic analysis, we found that nucleotide homology were respectively 94%、89%、89%、92%、93% and 92% when compared with mice, rats, cattle, horses and chimpanzees, and amino acids homology were respectively 98%、96%、96%、94%、98% and 97%. These results indicated that Wnt10b gene are highly conserved among different species. This gene encoded 394 amino acids, and contains conserve WNT1 functional domain and glycosylation site of Wnt family.
2. RT-PCR results of expression in different organs showed that Wnt10b express highest in the liver, and also expressed in heart, muscle, subcutaneous fat and pleen.
3. As the key factor of Wnt /β-catenin pathway , TCF4 full-length CDS sequence (2015 bp) was successfully cloned. submitted to GenBank, and gain a GenBank login number EU694100. Blast showed that homology of TCF4 were respectively94 %、90%、89%、93%、93% and 95% compared with mice, rats, monkeys, cows and horse and has been released, TCF4 genes than the right, followed by the homology of 94% and 90%, 89%, 93%, 93% and 95%. Bioinformatics analysis showed that open reading frames was located in site 1-1281.
4. Pig skeletal muscle cells were treated with Wnt /β-catenin signaling pathway activator LiCl(25mM). Results showed that activated Wnt /β-catenin signaling pathway could enhance the differentiation of skeletal myoblasts. Meanwhile we found thatβ-catenin protein expression gradually increased during the process of myoblast differentiation.
本试验以初生健康长白仔猪为试验动物,采用RT-PCR技术成功克隆猪Wnt10b基因与Wnt/β-catenin信号通路关键因子TCF4;分离培养骨骼肌肌卫星细胞,Wnt/β-catenin信号通路激活剂25mM LiCl处理,采用四甲基偶氮唑蓝(MTT)比色、HE染色、半定量RT-PCR和Western Blot等方法,研究Wnt/β-catenin信号通路对肌卫星细胞增殖分化的影响,并对其机理进行初步探讨。主要获得以下研究结果:
1.克隆了猪Wnt10b基因部分CDS序列1190bp,包含起始密码子ATG。向GenBank提交,获得登录号EU181371,并对其进行生物信息学分析,发现与GenBank上已公布的人、小鼠、大鼠、牛、马和黑猩猩核苷酸的同源性分别为94%、89%、89%、92%、93%和92%;氨基酸同源性分别为98%、96%、96%、94%、98%和97%。表明该基因在不同物种间具有很高的保守性。该段基因编码394个氨基酸,具有Wnt家族保守的WNT1功能结构域和糖基化位点。
2. RT-PCR检测Wnt10b在猪不同组织中的表达差异,结果表明该基因在猪肝脏中表达量最高,心脏、肌肉组织、皮下脂肪以及脾脏中也表达。
3.成功克隆Wnt/β-catenin通路关键因子TCF4基因全长CDS序列2015bp,提交GenBank获得登录号EU694100。与GenBank上已公布的人、小鼠、大鼠、猕猴、牛和马的TCF4基因进行比对,同源性依次为94 %、90%、89%、93%、93%和95%。生物信息学分析发现开放式阅读框位于1-1281位。
4. 25mM Wnt/β-catenin信号通路激活剂LiCl处理猪骨胳肌细胞,发现能促进骨骼肌细胞的成肌分化,而且发现随着成肌分化的进行,β-catenin蛋白的表达量逐渐上升。
In skeletal muscle tissues, myoblasts which are precursor cells of muscle tissue exist in the form of muscle satellite cells, and plays an important role in the skeletal muscle growth, development, injury and transplantation. It is practically significant to research on how to control the differentiation of muscle satellite cell effectively. Wnt protein is a kind of secreting protein that were encoded by the Wnt genes. After binding with its receptors located on the cell membrane through an auto/paracrine manner, wnt protein activate downstream cell signaling pathways,regulate the expression of target gene and early embryonic development, cell polarity and cell fate decision. In addition, Wnt signaling pathway is mainly involved in the process of cell proliferation, differentiation, polarization, apoptosis and anti-apoptosis during the embryonic development. What’s more, Wnt signaling pathway is necessary for the muscle formation process during the embryonic development, and the key regulatory factor of myoblast terminal differentiation and muscle satellite cell decision. Wnt10b can pecifically regulate adipogenesis potential of myogenesis and regenerated muscle, and activation of Wnt10b can inhibit adipocyte differentiation.
In this study, primary health Landrace pigs were adopted as experimental animals. Then Wnt10b gene and TCF4 which was key factor of Wnt/β-catenin signaling pathway were successfully cloned using RT-PCR technique. Skeletal muscle satellite cells were isolated, and treated with LiCl (25 mM) which was activator of Wnt/β-catenin signaling pathway, then a our-methyl-tetrazolium (MTT) colorimetric method, HE staining, HE staining, semi-quantitative RT-PCR and Western Blot methods was applied to study the effects of Wnt/β-catenin signaling pathway on muscle satellite cell proliferation and differentiation ,and discussed their mechanism. The following were the major findings:
1. 1190 bp of partially pig Wnt10b gene CDS sequence was Cloned, including start codon ATG. Submitted to GenBank, gain a login number EU181371. After an bioinformatic analysis, we found that nucleotide homology were respectively 94%、89%、89%、92%、93% and 92% when compared with mice, rats, cattle, horses and chimpanzees, and amino acids homology were respectively 98%、96%、96%、94%、98% and 97%. These results indicated that Wnt10b gene are highly conserved among different species. This gene encoded 394 amino acids, and contains conserve WNT1 functional domain and glycosylation site of Wnt family.
2. RT-PCR results of expression in different organs showed that Wnt10b express highest in the liver, and also expressed in heart, muscle, subcutaneous fat and pleen.
3. As the key factor of Wnt /β-catenin pathway , TCF4 full-length CDS sequence (2015 bp) was successfully cloned. submitted to GenBank, and gain a GenBank login number EU694100. Blast showed that homology of TCF4 were respectively94 %、90%、89%、93%、93% and 95% compared with mice, rats, monkeys, cows and horse and has been released, TCF4 genes than the right, followed by the homology of 94% and 90%, 89%, 93%, 93% and 95%. Bioinformatics analysis showed that open reading frames was located in site 1-1281.
4. Pig skeletal muscle cells were treated with Wnt /β-catenin signaling pathway activator LiCl(25mM). Results showed that activated Wnt /β-catenin signaling pathway could enhance the differentiation of skeletal myoblasts. Meanwhile we found thatβ-catenin protein expression gradually increased during the process of myoblast differentiation.
引文
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