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目的探讨上皮细胞条件培养液(epithelial cell conditioned medium,ECCM)体外诱导犬BMSCs向上皮细胞分化的可行性。方法成年健康雄性比格犬1只,体重10kg。于犬髂后上棘采用骨髓穿刺法取骨髓5mL,分离培养BMSCs。取犬口腔黏膜下组织,剪成约4mm×4mm大小,制备ECCM。取第2代BMSCs以2×104个/孔细胞密度接种,实验组于ECCM中培养,对照组于低糖DMEM完全培养基中培养。观察两组细胞形态特征,MTT法绘制生长曲线,诱导培养21d免疫组织化学染色鉴定上皮细胞特异性标志物——细胞角蛋白19(cytokeratin19,CK-19)和细胞角蛋白AE1/AE3,透射电镜观察细胞超微结构。结果倒置相差显微镜下见两组细胞均呈长梭形,均匀分布,折光性较强,增殖迅速。两组细胞生长曲线均呈“S”形,实验组生长曲线较对照组右移,提示ECCM对细胞增殖有抑制作用。实验组CK-19和细胞角蛋白AE1/AE3免疫组织化学染色均呈阳性反应,对照组呈阴性。透射电镜下实验组细胞胞浆内可见紧密连接。结论ECCM体外有诱导同种BMSCs向上皮细胞分化的作用。
Objective To investigate the feasibility of inducing the differentiation of canine BMSCs into epithelial cells in vitro by epithelial cell conditioned medium (ECCM). Methods One adult healthy beagle dog was born weighing 10kg. Bone marrow 5 mL was obtained by bone marrow aspiration in the posterior superior iliac spine of the dog, and BMSCs were isolated and cultured. Take dog oral submucosal tissue, cut into about 4mm × 4mm size, preparation of ECCM. The 2nd generation BMSCs were inoculated at a cell density of 2 × 10 4 cells / well. The experimental group was cultured in ECCM and the control group was cultured in low glucose DMEM complete medium. The morphological characteristics of the two groups were observed. The growth curve was drawn by MTT method. The epithelial cell specific markers such as cytokeratin 19 (CK-19) and cytokeratin AE1 / AE3 were identified by immunohistochemical staining on day 21. Transmission electron microscopy Observation of cell ultrastructure. Results Under inverted phase contrast microscope, both cells showed long fusiform shape with uniform distribution, strong refraction and rapid proliferation. The cell growth curve of both groups showed “S ” shape, and the growth curve of the experimental group shifted to the right of the control group, suggesting ECCM has an inhibitory effect on cell proliferation. Immunohistochemical staining of CK-19 and cytokeratin AE1 / AE3 in the experimental group was positive, but negative in the control group. Transmission electron microscopy of the experimental group showed tight junctions in the cytoplasm. Conclusion ECCM induces the differentiation of allogenic BMSCs into epithelial cells in vitro.