Abstract：Muscle satellite cells are potential myogenic cells capable of self-renewal and with potential multi-directional differentiation. Under normal circumstances, these cells are in resting state, but they can proliferate, differentiate and integrate into myotubes and form muscle cells. However, in vivo, these mesoderm originated satellite cells can not trans-differentiate into endoderm-derived insulin-producing cells. In this study, a muscle satellite cell line was isolated, cultured, identified and established from a bovine (Bos taurus) fetal muscle tissues. The muscle satellite cells were then induced to differentiate into insulin-producing cells. During pancreatic induction process, the genes associated with pancreatic development such as pancreatic and duodenal homeobox 1 (PDX1), neurogenin 3 (NGN3), Amylase and Insulin (INS) were analyzed. PDX1, an important transcription factor in differentiation of pancreatic islets, started to express after induction 2 d, reached to the highest level after 3 d, and kept lowest level after 4 d. NGN3 was an important transcription factor in the formation of endocrine cells, and was a key gene that controlled insulin secretion to extracellular, which the mRNA started to express after induction 3 d, reached to the highest level after 4 d and then declined. As a pancreatic extracellular secretion associated gene, Amylase mRNA appeared after 6 d and reached to the maximum after 8 d. INS expressed from 1st day and reached to the maximum at 11~12 d. Enzyme-linked immunosorbent assay (ELISA) of the culture media showed that the concentration of insulin secreted by the cultured cells reached to the maximum at 11~12 d. The insulin secretion was significantly increased in the glucose added culture medium, and glucose concentration regulated insulin secretion in the cultured cells, which was similar to adult islet fuction. In conclusion, the bovine muscle satellite cells could be trans-differentiated into pancreatic cells, and make response to glucose stimulation. The study provides basic data for further research and clinical application.
[1]边艳超, 何晓红, 牧仁, 等.牛胚胎骨骼肌卫星细胞的分离, 培养及诱导分化[J].细胞与分子免疫学杂志, 2013, 29(11):1196-1199[2]宫佳琦, 李瑞文, 吕彦红, 等.草鱼 基因的克隆及其组织表达谱分析[J].中国农学 通报, 2012, 28(29):125-130[3]郭欣, 李国立, 黄跃南, 等.成体胰腺干细胞基础研究进展[J].世界华人消化杂志, 2006, 14(4):422-428[4]刘月光, 史新娥, 沈清武, 等.利用单根肌纤维法分离和培养猪骨骼肌卫星细胞及其成肌诱导分化[J].农业生物技术学报, 2011, 19(5):856-863[5]杨柏梁, 孙庆, 郭丽, 等.骨髓间充质干细胞诱导分化为胰岛样细胞及其功能的研究[J].中国卫生工程学, 2011, 10(4):303-306[6]Ber I, Shternhall K, Perl S, et al.Functional,persistent,and extended liver to pancreas transdifferentiation[J].Journal of Biological Chemistry, 2003, 278(34):31950-31957[7]Benchabane H, Xin N, Tian A, et al.JerkyEarthbound facilitates cell-specific WntWingless signalling by modulating β-catenin-TCF activity[J].The EMBO Journal, 2011, 30(8):1444-1458[8]Chen Y H, Tsai H J.Myogenic regulatory factors Myf5 and Mrf4 of fish: current status and perspective[J].Journal of Fish Biology, 2008, 73(8):1872-1890[9]Desgraz R，Herrera P L.Pancreatic neurogenin 3-expressing cells are unipotent islet precursors[J].Development, 2009, 136(21):3567-3574[10]Holst D, Luquet S, Kristiansen K, et al.Roles of peroxisome proliferator-activated receptors delta and gamma in myoblast transdifferentiation[J].Experimental Cell Research, 2003, 288(1):168-176[11]Jensen J, Heller R, Funder-Nielsen T, et al.Independent development of pancreatic alpha-and beta-cells from neurogenin3-expressing precursors: A role for the notch pathway in repression of premature differentiation[J[J].Diabetes, 2000, 49( (2)):163-176[12]Jiang F X，Morahan G.Pancreatic stem cells: from possible to probable[J].Stem Cell Rev, 2012, 8(3):647-657[13]Kajiyama H, Hamazaki T S, Tokuhara M, et al.Pdx1-transfected adipose tissue-derived stem cells differentiate into insulin-producing cells in vivo and reduce hyperglycemia in diabetic mice[J].International Journal of Developmental Biology, 2010, 54(4):699-705[14]Karlsson O, Edlund T, Moss JB, et al.A mutational analysis of the insulin gene transcription control region: expression in beta cells is dependent on two related sequences within the enhancer[J].Proceedings of the National Academy of Sciences, 1987, 84(24):8819-8823[15]Kodama S, Kuhtreiber W, Fujimura S, et al.Islet regeneration during the reversal of autoimmune diabetes in NOD mice[J].[J].Science, 2003, 30(2):1223-1227[16]Magee TR, Artaza JN, Ferrini MG, et al.Myostatin short interfering hairpin RNA gene transfer increases skeletal muscle mass[J].The Journal of Gene Medicine, 2006, 8(9):1171-1181[17]Miyatsuka T, Kosaka Y, Kim H, et al.Neurogenin3 inhibits proliferation in endocrine progenitors by inducing Cdkn1a.[J].Proc Natl Acad Sci, 2011, 108(1):185-190[18]Rocheteau P, Gayraud-Morel B, Siegl-Cachedenier I, et al.A subpopulation of adult skeletal muscle stem cells retains all template DNA strands after cell division[J].Cell, 2012, 148(1):112-125[19]Ramiya VK, Maraist M, Arfors KE, et al.Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells[J].Nature Medicine, 2000, 6(3):278-282[20]Soria B, Roche E, Berna G, et al.Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice.[J].Diabetes, 2000, 49(1):157-162[21]Samaras SE, Zhao L, Means A, et al.The islet β cell-enriched RIPE3b1Maf transcription factor regulates pdx-1 expression[J].Journal of Biological Chemistry, 2003, 278(14):12263-12270[22]TangDQ, CaoLZ, Burkhardt BR, et al.Invivoandinvitro Characterization of insulin producing cells obtained frommurine bone marrow[J].Diabetes, 2004, 53(7):1721-1732[23]Wang S, Jensen JN, Seymour PA, et al.Sustained Neurog3 expression in hormone-expressing islet cells is required for endocrine maturation and function .[J].Proc Natl Acad Sci USA, 2009, 106(24):9715-9720[24]Wada MR, Inagawa-Ogashiwa M, Shimizu S, et al.Generation of different fates from multipotent muscle stem cells[J].Development, 2002, 129(12):2987-2995[25]Zhang D, Jiang W, Liu M, et al.Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells[J].Cell Research, 2009, 19(4):429-438
