| 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.
|
PDF Full Text Request