犬骨髓多能成体祖细胞的体外培养、鉴定及向平滑肌样细胞的诱导分化

Culture and identification of canine bone marrow-derived multipotent adult progenitor cells and their differentiation into smooth muscle-like cells

目的:探讨犬骨髓多能成体祖细胞(MAPC)的体外培养条件、生物学特性及其向平滑肌样细胞分化的可能性。方法:观察体外培养犬骨髓多能成体祖细胞的形态、生长情况,检测其表面标志,利用流式细胞技术检测MAPC的生长周期及特异表面标志表达率;并以分化培养基诱导培养,采用RT-PCR方法检测骨髓多能成体祖细胞OCT-4及分化细胞SM-MHC (smooth muscle cells-myosin heavy chain)表达;检测分化细胞有无平滑肌细胞表面标志的表达。结果:光镜下观察犬骨髓多能成体祖细胞体积偏大,长多角型,数个细长伪足,细胞核大,胞质丰富,细胞增殖能力旺盛;表达表面标志CD13和SSEA-1.不表达CD44、CD45、CD133和MHCⅡ;RT-PCR结果显示表达OCT-4。诱导分化后,分化细胞表达平滑肌细胞表面标志——α^- SMA、calponin、SM-MHC;RT-PCR结果显示表达SM-MHC。结论:体外成功培养犬骨髓多能成体祖细胞,具有与文献报道类似的生物学特征;MAPC在一定诱导条件下具有向平滑肌样细胞分化的潜能。

Objective： To study the culture condition and biological characteristics of canine muhipotent adult progenitor cells（MAPC） and their potential to differentiate into smooth muscle-like cells in vitro. Methods： The morphology and growth of in vitro cultured MAPC were observed and the surface marker of MAPC was detected. The cell cycle and phenotype of MAPC were determined by FACS. RT-PCR was used to examine the expression of OCT-4 in MAPC and smooth muscle cells-myosin heavy chain （SM-MHC） in the differentiated cells. The surface markers of smooth muscle cells were detected in the differentiated cells. Results： Under microscope, MAPCs were polygon and had large nuclei, muhi-parapodiums and affluent cytoplasma. The cells also showed strong proliferation ability and were positive of CD13, SSEA-1 and negative of CD44, CD45, CD133, and MHCⅡ. RT-PCR showed expression of OCT-4. The cells differentiated from MAPC expressed the surface markers of smooth muscle cells, including a-SMA, calponin and SM-MHC. RT-PCR demonstrated the expression of SM- MHC. Conclusion： We have successfully cultured canine MAPC in vitro, which has similar biological characteristics as reported previously. MAPC has the potential to differentiate into smooth muscle-like cells under certain physiological condition in vitro.