SFRP2对脂肪干细胞成骨分化的作用研究

›› 2018, Vol. 9 ›› Issue (3): 120-124.

SFRP2对脂肪干细胞成骨分化的作用研究

杨昊清¹,范志朋²

1. 首都医科大学附属北京口腔医院
2. 首都医科大学附属北京口腔医院研究所

收稿日期:2018-03-27 修回日期:2018-07-12 出版日期:2018-09-25 发布日期:2018-09-29
通讯作者: 范志朋 E-mail:wang54384@163.com
基金资助:
北京市自然科学基金

SFRP2 promoted osteogenic differentiation of adipose-derived stem cells

Hao-Qing YANG¹, ²

1. ,Capital Medical University School of Stomatology
2. Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, School of Stomatology, Capital Medical University

Received:2018-03-27 Revised:2018-07-12 Online:2018-09-25 Published:2018-09-29
Supported by:
Beijing Natural Science Foundation

摘要/Abstract

摘要： 目的：研究分泌型卷曲相关蛋白2（SFRP2）对脂肪干细胞（ADSC）成骨分化的影响。。方法：利用重组慢病毒载体的SFRP2 shRNA基因敲除ADSC中SFRP2进行丧失性功能研究；利用HA-SFRP2逆转录病毒载体过表达SFRP2进行获得性功能研究；定量RT-PCR和Western Blot检测基因表达；碱性磷酸酶活性（ALP）测定、茜素红染色、钙离子定量以及定量RT-PCR检测脂肪干细胞的体外成骨分化能力。结果：定量RT-PCR和Western Blot显示SFRP2sh可以在ADSC中有效的抑制SFRP2的表达；ALP、茜素红和钙离子定量结果显示敲除SFRP2能明显抑制ADSC体外成骨分化能力；定量RT-PCR结果显示敲除SFRP2抑制骨涎蛋白（BSP）、Osterix（Osx）和Runt相关转录因子2（RUNX2）的表达；定量RT-PCR和Western Blot显示HA-SFRP2可以在ADSC中有效的过表达；ALP、茜素红和钙离子定量结果显示过表达SFRP2能明显促进ADSC体外成骨分化能力；定量RT-PCR显示过表达SFRP2明显促进BSP的表达。结论：SFRP2促进脂肪干细胞的体外成骨分化功能。

Abstract: Objective：To investigate the effect of SFRP2 on osteogenic differentiation potential of adipose-derived stem cells (ADSC). Methods：Lentiviral SFRP2 shRNA was used to silence the expression of SFRP2. Retroviral HA-SFRP2 was used to over-express the SFRP2 in ADSC; real-time RT-PCR and Western Blot were used to detect the expression of SFRP2. Alkaline phosphatase activity, alizarin red staining, mineralization and real-time RT-PCR were used to detect the osteogenic differentiation of adipose-derived stem cells. Results：Real-time RT-PCR and Western Blot results showed that SFRP2sh effectively reduced the expression of SFRP2 in ADSC. Depletion of SFRP2 reduced alkaline phosphatase, mineralization and bone sialoprotein (BSP), Osterix (Osx) and runt-related transcription factor-2 (RUNX2) in ADSC. Real-time RT-PCR and Western Blot showed that HA-SFRP2 can be effectively overexpressed in ADSC. The results of alkaline phosphatase, alizarin red and calcium qualitative analysis results showed that the over expressed SFRP2 in ADSC could significantly promote the osteogenic differentiation of ADSC. Real-time RT-PCR results showed that the SFRP2 significantly promoted the expression of BSP. Conclusions：SFRP2 can promote osteogenic differentiation of adipose stem cells and prove that SFRP2 is the key gene for osteogenic differentiation of ADSC.

杨昊清范志朋. SFRP2对脂肪干细胞成骨分化的作用研究[J]. , 2018, 9(3): 120-124.

Hao-Qing YANG. SFRP2 promoted osteogenic differentiation of adipose-derived stem cells[J]. , 2018, 9(3): 120-124.

参考文献

[1]Mochizuki T, Muneta T, Sakaguchi Y, et al.Higher chondrogenic potential of fibrous synovium- and adipose synovium-derived cells compared with subcutaneous fat-derived cells: distinguishing properties of mesenchymal stem cells in humans[J].Arthritis Rheum, 2006, 54(3):843-853
[2]Lee JA, Parrett BM, Conejero JA, et al.Biological alchemy: engineering bone and fat from fat-derived stem cells[J].Ann Plast Surg, 2003, 50(6):610-617
[3]Fink T, Rasmussen JG, Emmersen J, et al.Adipose-derived stem cells from the brown bear (Ursus arctos) spontaneously undergo chondrogenic and osteogenic differentiation in vitro[J].Stem Cell Research, 2011, 7(1):89-95
[4]Safford KM, Hicok KC, Safford SD, et al.Neurogenic differentiation of murine and human adipose-derived stromal cells[J].Biochem Biophys Res Commun, 2002, 294(2):371-379
[5]Ravichandran R, Venugopal JR, Mueller M, et al.Buckled structures and 5-azacytidine enhance cardiogenic differentiation of adipose-derived stem cells[J].Nanomedicine, 2013, 8(12):1985-1997
[6]De Ugarte DA, Morizono K, Elbarbary A, et al.Comparison of multi-lineage cells from human adipose tissue and bone marrow[J].Cells Tissues Organs, 2003, 174(3):101-109
[7]Gir P, Oni G, Brown SA, et al.Human adipose stem cells: current clinical applications[J].Plast Reconstr Surg, 2012, 129(6):1277-1290
[8]Crowley RK, O’Reilly MW, Bujalska IJ, et al.SFRP2 Is Associated with Increased Adiposity and VEGF Expression[J].PLoS One, 2016, 11(9):e0163777-
[9]Lin H, Angeli M, Chung KJ, et al.sFRP2 activates Wnt/β-catenin signaling in cardiac fibroblasts: differential roles in cell growth, energy metabolism, and extracellular matrix remodeling[J].Am J Physiol Cell Physiol, 2016, 311(5):C710-C719
[10]Majchrzak-Celińska A, S?ocińska M, Barciszewska AM, et al.Wnt pathway antagonists, SFRP1, SFRP2, SOX17, and PPP2R2B, are methylated in gliomas and SFRP1 methylation predicts shorter survival[J].J Appl Genet, 2016, 57(2):189-197
[11]Yu G, Wang J, Lin X, et al.Demethylation of SFRP2 by histone demethylase KDM2A regulated osteo-/dentinogenic differentiation of stem cells of the apical papilla[J].Cell Prolif, 2016, 49(3):330-340
[12]Tirkkonen L, Haimi S, Huttunen S, et al.Osteogenic medium is superior to growth factors in differentiation of human adipose stem cells towards bone-forming cells in 3D culture[J].Eur Cell Mater, 2013, 25(11):144-158
[13]Quarto N, Wan DC, Longaker MT.Molecular mechanisms of FGF-2 inhibitory activity in the osteogenic context of mouse adipose-derived stem cells (mASCs)[J].Bone, 2008, 42(6):1040-1052
[14]Elabd C, Chiellini C, Massoudi A, et al.Human adipose tissue-derived multipotent stem cells differentiate in vitro and in vivo into osteocyte-like cells[J].Biochem Biophys Res Commun, 2007, 361(2):342-348
[15]Rider DA, Dombrowski C, Sawyer AA, et al.Autocrine fibroblast growth factor 2 increases the multipotentiality of human adipose-derived mesenchymal stem cells[J].Stem Cells, 2008, 26(6):1598-1608
[16]Ling L, Nurcombe V, Cool SM.Wnt signaling controls the fate of mesenchymal stem cells[J].Gene, 2009, 433(1/2):1-7
[17]Alfaro MP, Vincent A, Saraswati S, et al.sFRP2 suppression of bone morphogenic protein (BMP) and Wnt signaling mediates mesenchymal stem cell (MSC) self-renewal promoting engraftment and myocardial repair[J].J Biol Chem, 2010, 285(46):35645-35653
[18]Jin L, Yu C, Yu G, et al.SFRP2 enhances the osteogenic differentiation of apical papilla stem cells by antagonizing the canonical WNT pathway[J].Cell Mol Biol Lett, 2017, 22:14-
[19]Paz J, Wade K, Kiyoshima T, et al.Tissue- and bone cell-specific expression of bone sialoprotein is directed by a 9.0 kb promoter in transgenic mice[J].Matrix Biol, 2005, 24(5):341-352
[20]Malaval L, Wade-Guéye NM, Boudiffa M, et al.Bone sialoprotein plays a functional role in bone formation and osteoclastogenesis[J].J Exp Med, 2008, 205(5):1145-1153