EFFECT OF WNT6 IN PROLIFERATION, DIFFERENTIATION, AND MIGRATION OF BONE MARROW MESENCHYMAL STEM CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

CAOYujing ¹ ,  LVCuitian ²

1. Department of Traumatic Orthopedics, Henan Province Hospital of Traditional Chinese Medicine, Zhengzhou Henan, 450002, P. R. China;
2. Basic Medical College, Henan University of Traditional Chinese Medicine;

Corresponding?author：

LVCuitian, Email: submission029@gmail.com

Keywords：

WNT6; Bone marrow mesenchymal stem cells; Cell proliferation; Cell migration; Osteogenic

DOI：

10.7507/1002-1892.20150019

Video：

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Objective To explore the potential role of WNT6 in the proliferation, differentiation, and migration of bone marrow mesenchymal stem cells (BMSCs). Methods Mouse BMSCs were cultured to the cell fusion of 30%-50%, and divided into different groups. WNT6 knockdown included 3 experiment groups:cells transfected with WNT6 specific short hairpin RNA (shRNA) (group A1), cells transfected with control shRNA group (group B1), and nontransfected cells (group C1). WNT6 over-expression included 3 groups:cells transfected with WNT6 recombinant plasmid (group A2), cells transfected with blank vector (group B2), and non-transfected cells (group C2). After transfection, the stably transfected cells were cultured for 48 hours. Cell morphology was observed under inverted microscope; real-time fluorescent quantitative PCR was used to analyze WNT6 mRNA levels; Western blot was used to detect WNT6 and Ki67 protein expressions; cell proliferation was assayed by MTT method, and cell migration was detected by Transwell assay. After cells were cultured in osteogenic differentiation medium for 12 days, the alkaline phosphatase (ALP) activity and calcium deposits were detected by biochemical determination. Results The inverted microscope observation showed that the cell morphology were similar among groups A1, B1, C1, and A2, B2, C2. The WNT6 mRNA and protein levels, Ki67 protein level, cell proliferation, cell migration, ALP activity, and calcium deposition in group A1 were all significantly lower than those in groups B1 and C1 (P<0.05), but there was no significant difference between groups B1 and C1 (P>0.05). On the contrary, the above indexes in group A2 were all significantly higher than those in groups B2 and C2 (P<0.05), but no significant difference was shown between groups B2 and C2 (P>0.05). Conclusion WNT6 can promote the proliferation and migration, as well as can enhance osteogenic differentiation ability in mouse BMSCs.

Citation： CAOYujing, LVCuitian. EFFECT OF WNT6 IN PROLIFERATION, DIFFERENTIATION, AND MIGRATION OF BONE MARROW MESENCHYMAL STEM CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(1): 92-96. doi: 10.7507/1002-1892.20150019 Copy

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2.	曹振昊, 田云虎, 馬璐璐, 等. 慢性壓迫背根神經節大鼠模型背根神經節Wnt5a的表達變化. 中國修復重建外科雜志, 2014, 28(11):1392-1396.
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13.	Tashiro K, Kondo A, Kawabata K, et al. Efficient osteoblast differentiation from mouse bone marrow stromal cells with polylysin-modified adenovirus vectors. Biochem Bioph Res Commun, 2009, 379(1):127-132.
14.	Chen KY, Chung CM, Chen YS, et al. Rat bone marrow stromal cells-seeded porous gelatin/tricalcium phosphate/oligomeric proanthocyanidins composite scaffold for bone repair. J Tissue Eng Regen Med, 2013, 7(9):708-719.
15.	Wang CZ, Fu YC, Jian SC, et al. Synthesis and characterization of cationic polymeric nanoparticles as simvastatin carriers for enhancing the osteogenesis of bone marrow mesenchymal stem cells. J Colloid and Interf Sci, 2014, (432):190-199.
16.	Cawthorn WP, Bree AJ, Yao Y, et al. Wnt6, Wnt10a and Wnt10b inhibit adipogenesis and stimulate osteoblastogenesis through a β-catenin-dependent mechanism. Bone, 2012, 50(2):477-489.
17.	Zhang M, Sun L, Wang X, et al. Activin B promotes BMSCmediated cutaneous wound healing by regulating cell migration via the JNK-ERK signaling pathway. Cell Transplant, 2014, 23(9):1061-1073.
18.	Sangani R, Pandya CD, Bhattacharyya MH, et al. Knockdown of SVCT2 impairs in-vitro cell attachment, migration and wound healing in bone marrow stromal cells. Stem Cell Res, 2014, 12(2):354-363.
19.	Handrigan GR, Richman JM. Autocrine and paracrine Shh signaling are necessary for tooth morphogenesis, but not tooth replacement in snakes and lizards (Squamata). Dev Biol, 2010, 337(1):171-186.
20.	Li R, Wang C, Tong J, et al. WNT6 promotes the migration and differentiation of human dental pulp cells partly through c-Jun N-terminal kinase signaling pathway. J Endod, 2014, 40(7):943-948.
21.	Wang C, Ren L, Peng L, et al. Effect of Wnt6 on human dental papilla cells in vitro. J Endod, 2010, 36(2):238-243.

1. 李萍華, 劉鈺瑜, 崔燎. 骨髓間充質干細胞成脂和成骨分化過程中Wnt信號通路的調控效應. 中國組織工程研究與臨床康復, 2010, 14(10):1749-1754.
2. 曹振昊, 田云虎, 馬璐璐, 等. 慢性壓迫背根神經節大鼠模型背根神經節Wnt5a的表達變化. 中國修復重建外科雜志, 2014, 28(11):1392-1396.
3. Niehrs C, Acebron SP. Mitotic and mitogenic Wnt signalling. EMBO J, 2012, 31(12):2705-2713.
4. Tan SH, Senarath-Yapa K, Chung MT, et al. Wnts produced by Osterix-expressing osteolineage cells regulate their proliferation and differentiation. P Natl Acad Sci USA, 2014, 111(49):E5262-5271.
5. Hwang JT, Kelly GM. GATA6 and FOXA2 regulate Wnt6 expression during extraembryonic endoderm formation. Stem Cells Dev, 2012, 21(17):3220-3232.
6. Lavery DL, Martin J, Turnbull YD, et al. Wnt6 signaling regulates heart muscle development during organogenesis. Dev Biol, 2008, 323(2):177-188.
7. Wang Q, Lu J, Zhang S, et al. Wnt6 is essential for stromal cell proliferation during decidualization in mice. Biol Reprod, 2013, 88(1):5.
8. Zhou H, Yang X, Wang N, et al. Tigogenin inhibits adipocytic differentiation and induces osteoblastic differentiation in mouse bone marrow stromal cells. Mol Cell Endocrinol, 2007, 270(1-2):17-22.
9. Witte F, Dokas J, Neuendorf F, et al. Comprehensive expression analysis of all Wnt genes and their major secreted antagonists during mouse limb development and cartilage differentiation. Gene Expr Patterns, 2009, 9(4):215-223.
10. Mamo S, Gal AB, Bodo S, et al. Quantitative evaluation and selection of reference genes in mouse oocytes and embryos cultured in vivo and in vitro. BMC Dev Biol, 2007, 7:14.
11. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative CT method. Nat Protoc, 2008, 3(6):1101-1108.
12. 廖清船, 肖洲生, 秦艷芳, 等. 植物雌激素金雀異黃酮通過p38MAPK通路促進骨髓間充質干細胞向成骨細胞分化. 中國藥理學通報, 2006, 22(6):683-687.
13. Tashiro K, Kondo A, Kawabata K, et al. Efficient osteoblast differentiation from mouse bone marrow stromal cells with polylysin-modified adenovirus vectors. Biochem Bioph Res Commun, 2009, 379(1):127-132.
14. Chen KY, Chung CM, Chen YS, et al. Rat bone marrow stromal cells-seeded porous gelatin/tricalcium phosphate/oligomeric proanthocyanidins composite scaffold for bone repair. J Tissue Eng Regen Med, 2013, 7(9):708-719.
15. Wang CZ, Fu YC, Jian SC, et al. Synthesis and characterization of cationic polymeric nanoparticles as simvastatin carriers for enhancing the osteogenesis of bone marrow mesenchymal stem cells. J Colloid and Interf Sci, 2014, (432):190-199.
16. Cawthorn WP, Bree AJ, Yao Y, et al. Wnt6, Wnt10a and Wnt10b inhibit adipogenesis and stimulate osteoblastogenesis through a β-catenin-dependent mechanism. Bone, 2012, 50(2):477-489.
17. Zhang M, Sun L, Wang X, et al. Activin B promotes BMSCmediated cutaneous wound healing by regulating cell migration via the JNK-ERK signaling pathway. Cell Transplant, 2014, 23(9):1061-1073.
18. Sangani R, Pandya CD, Bhattacharyya MH, et al. Knockdown of SVCT2 impairs in-vitro cell attachment, migration and wound healing in bone marrow stromal cells. Stem Cell Res, 2014, 12(2):354-363.
19. Handrigan GR, Richman JM. Autocrine and paracrine Shh signaling are necessary for tooth morphogenesis, but not tooth replacement in snakes and lizards (Squamata). Dev Biol, 2010, 337(1):171-186.
20. Li R, Wang C, Tong J, et al. WNT6 promotes the migration and differentiation of human dental pulp cells partly through c-Jun N-terminal kinase signaling pathway. J Endod, 2014, 40(7):943-948.
21. Wang C, Ren L, Peng L, et al. Effect of Wnt6 on human dental papilla cells in vitro. J Endod, 2010, 36(2):238-243.

Chinese Journal of Reparative and Reconstructive Surgery

EFFECT OF WNT6 IN PROLIFERATION, DIFFERENTIATION, AND MIGRATION OF BONE MARROW MESENCHYMAL STEM CELLS

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