Abstract：In order to isolate the bone marrow mesenchymai stem cells (BMSCs) of dog (Canis lupus) and induce the BMSCs to differentiate into osteoblastic cells, the BMSCs were isolated in vitro by whole bone marrow adherent method. After proliferation into full confluency, the BMSCs were subcultured. During proliferation, cell growth curve assay was performed in different passages. Pluripotent markers (SRY-related high-monility-group (HMG)-box protein-2 (SOX2), octamer-binding transcription factor-4 (OCT4) and NANOG) and osteoblast specific markers (osteocalcin (OCN) and osteonectin (ONN)) were defected by qRT-PCR in undifferentiated cells and cells induced differentiation for 10 and 21 d. Alkaline phosphatase (ALP) and alizarin red were defected by staining in undifferentiated cells and cells induced differentiation for different days. The results showed that the growth curve of undifferentiated cells in p5 and p10 were similar. However, the proliferation rate of cells in p20 was significantly slower than that of cells in p5 and p10. Before differentiation, the pluripotent genes such as OCT4 and SOX2, but not NANOG expressed in the BMSCs. The low expression levels of OCT4 and SOX2 could also be detected after induced differentiation for 10 days. However, after induced differentiation for 21 days, there was no expression of OCT4 and SOX2 in differentiated cells. For osteoblast specific markers, the expression levels of OCN and ONN increased along with induced differentiation. Alkaline phosphatase activity of differentiated cells significantly increased after induced for 7 days. Calcified nodules could be observed and stained red after induced for 21 days, and the number of calcified nodules increased along with induced differentiation. These results indicated that BMSCs of dog could be isolated and induced differentiation into mature osteoblasts successfully in vitro, which offer a new approach to the clinical treatment of severe fractures and osteodystrophic arthropathy of the pet dogs.
参考文献Schwartz RE, Reyes M, Koodie L, et al.2002. Multipotent adult progenitor cells from bone m arrow differentiate in to functional hepatocyte-like cells [J]. J Chin Invest, 109(10): 1291- 1302Spees JL, Olson SD, Ylostalo J, et al.2003. Differentiation, cell fusion, and nuclear fusion during ex vivo repair of epithelium by hum an adult stem cells from bone marrow stroma [J] . Proc Natl Acad Sci U S A, 100(5): 2397 - 2402 Di Nicola M, Carlosella C, Magni M, et al. 2002. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli[J]. Blood, 99(10):3838-3843.Le Blanc K, Tammik L, Sundberg B et al.2003. Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex[J]. Scand J Immunol, 57(1):11-20.Bruder SP, Kraus KH, Goldberg VM, et al.1998. The effect of implants loaded with autologous mesenchymal stem cells on the healing of canine segmental bone defects[J]. J Bone Joint Surg Am, 80(7): 985-996.Frosch KH, Drengk A, Krause P, et al.2006. Stem cell-coated titanium implants for the partial joint resurfacing of the knee[J]. Biomaterials, 27(12): 2542-2549.Quarto R, Mastrogiacomo M, Cancedda R, et al.2001. Repair of large bone defects with the use of autologous bone marrow stromal cells [J]. N Engl J Med, 344(5):385-386.Morishita T, Honoki K, Ohgushi H, et al.2006. Tissue engineering approach to the treatment of bone tumors: three cases of cultured bone grafts derived from patients' mesenchymal stem cells [J]. Artif Organs, 30(2): 115-118.Adewumi O, Aflatoonian B, Ahrlund-Richter L, et al.2007. Characterization of human embryonic stem cell lines by the international stem cell initiative[J]. Nature Biotechnology, 25(7): 803-816. Boiani M, Scholer H R.2005. Regulatory networks in embryo-derived pluripotent stem cells[J]. Nature Reviews Molecular Cell Biology, 6(11): 872-884.Yamanaka S, Li JL,KaniaG, et al.2008. Pluripotency of embryonic stemcells[J].Cell andTissue Research, 331(1): 5-22.Schoenhals M, Kassambara A, De Vos J, et al. 2009. Embryonic stem cell markers expression in cancers[J]. Biochemical and Biophysical Research Communications, 383(2): 157-162.丛姗, 宋瑾, 张惠娟,等. 2015. 人羊膜间充质干细胞(hAMSCs)的分离体外培养及诱导分化,农业生物技术学报, 23(1): 20~31.（Cong Shan, Song Jin, Zhang Hui-Juan et al. 2015. Isolation and Culture of Human Amniotic Mesenchymal Stem Cells (hAMSCs) and Its Differentiation Potential In vitro . Journal of Agricultural Biotechnology, 23(1): 20~31)惠慧,罗聪, 毕杨. 2014. 肝细胞生长因子促进骨形态发生蛋白9 诱导小鼠骨髓间充质干细胞C3H10 成骨分化的研究[J].第三军医大学学报,36(23):2080-2084.( Hui Hui，Luo Cong，Bi Yang. 2014. HGF promotes osteogenic differentiation in mesenchymal stem cells induced by bone morphogenetic protein 9[J]. J Third Mil Med Univ, 36(23): 2080-2084颜春鲁, 李盛华, 周明旺,等.2014. 加味左归丸诱导骨髓间充质干细胞骨向分化的实验研究[J], 中国骨质疏松杂志, 2014, 20(10): 1154-1158.( Yan Chun lu，Li Sheng hua ，Zhou Ming wang et al.2014. An experimental study of the osteoblast differentiation from mesenchymal stem cells by Zuogui pill induction [J]. Chin J Osteoporos,20(10): 1154-1158)Owe TA, Aronow M, Shalhoub V, et al.1990. Progressive development of rat osteoblast phenotypein vitro: reciprocal relationship in expression of genes associated with osteoblast proliferation and differentiation during formation of bone ECM [J]. J Cell Physiol, 143(3) : 420-425．
