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rspo2 promotes embryonic stem cells to differentiate towards smooth muscle cells through lgr5/β-catenin pathway

中国病理生理杂志 2016年第8期32卷 1531-1532页

作者： WU Yu-tao YANG Mei WANG Jing YANG Feng DAI Bing WANG Wei-na CHEN Ting ZHOU Yi-jiang ZHANG Li Department of Cardiology First Affiliated HospitalSchool of MedicineZhejiang University

AIM: R-spondin 2( rspo2),one member of R-spondin family which contains four secreted proteins,plays an important role in skeletal muscle development. However,the impact of rspo2 on vascular smooth muscle cell( SMC) differentiation is little *** study aims at revealing the role and mechanism of rspo2 on SMC differentiation from embryonic stem cells( ESCs). METHODS:A well-established model for studying SMC differentiation from ESCs were used,in which mouse embryonic stem cells( ES-D3) were seeded on collagen IV-coated asks and cultured in differentiation medium( DM) for 2,4,6 and 8 days. smooth muscle specific markers,including α-smooth muscle actin( α-SMA),SM22 and smooth muscle myosin heavy chain( SM-MHC),were detected to insure the successful model by q RT-PCR and Western blot. After 3-day pre-differentiation,ESCs were treated with recombinant rspo2 protein,overexpression plasmid or sh RNA plasmid for 96 h,and the m RNA and protein expression of smooth muscle markers was detected. To explore the role of rspo2 on SMC differentiation in vivo,3-day predifferentiated ESCs(106in 50 μLα-MEM) incubated with rspo2-overexpression plasmid were mixed with 50 μL of Matrigel( Becton Dickinson Labware) and then subcutaneously injected into C57 BL /6J mice. After 12 days,mice were sacrificed and the implants were harvested for immunofluorescence staining,q RT-PCR and Western blot. Furthermore,electrophoretic mobility shift assay( EMSA),chromatin immunoprecipitation assay( Ch IP) and luciferase reporter assay were performed to investigate the transcriptional activity of SMC differentiation related transcription factors,including serum response factor( SRF),myocardin( MYO),myocyte-specific enhancer factor 2C( MEF-2C). Involvement of rspo2 receptor,leucine-rich repeat-containing,G-protein-coupled receptors( lgr) 4,5,6,and β-catenin pathway during rspo2-induced MSC differentiation were also uncovered by overexpression or inhibition of the respective protein. RESULTS: Our resu

关键词： stem rspo2 promotes embryonic stem cells to differentiate towards smooth muscle cells through lgr5 catenin pathway

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