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目的观察犬骨髓间叶干细胞的体外多向分化潜力,为损伤组织的干细胞移植修复提供理论基础。方法利用梯度-贴壁筛选法分离、培养与扩增骨髓间叶干细胞。利用化学诱导剂5-氮胞苷(20滋mol/L),血管内皮细胞生长因子(VEGF10ng/ml),成骨细胞诱导剂(地塞米松10nMol/L,抗坏血酸0.05mMol,茁甘油磷酸钠10mMol/L)以及成脂肪细胞诱导剂(1-甲基3-异丁基黄嘌呤0.5mmol/L,地塞米松1滋mol/L,胰岛素10mg/L,消炎痛100mmol/L),分别定向诱导骨髓间叶干细胞分化为心肌细胞、血管内皮细胞、成骨细胞与脂肪细胞。再应用细胞形态观察、免疫细胞化学染色及电镜对分化细胞进行鉴定。结果利用梯度-贴壁筛选法可以分离培养与扩增骨髓间叶干细胞。5-氮胞苷可诱导干细胞呈现肌管、肌丝、心房颗粒,且肌细胞特异性蛋白琢-actinin,Myosin,琢-Actin,TroponinI免疫染色阳性;VEGF可诱导骨髓间叶干细胞出现管网状、血管样及鹅卵石样结构,vWF免疫染色阳性;成骨细胞诱导剂可诱导分化细胞碱性磷酸酶阳性;成脂肪细胞诱导剂使分化细胞内出现脂肪滴,油红O染色示脂滴为橙红色。结论成年犬骨髓间叶干细胞在体外具备多向分化潜力。在化学或生物诱导剂的作用下,犬骨髓间叶干细胞能够定向分化为心肌细胞、血管内皮细胞、成骨细胞、脂肪细胞等多种间叶组织?
OBJECTIVE: To observe the potential of multidirectional differentiation of dog bone marrow mesenchymal stem cells in vitro and to provide a theoretical basis for stem cell transplantation of damaged tissues. Methods The bone marrow mesenchymal stem cells were isolated, cultured and expanded by gradient - adherent screening method. 5-azacytidine (20 μmol / L), vascular endothelial growth factor (VEGF 10 ng / ml), osteoblast induction agent (10 nMol / L dexamethasone, 0.05 mMol ascorbic acid, 10 mMol / L) and adipocyte induction agents (1-methyl 3-isobutylxanthine 0.5 mmol / L, dexamethasone 1 mol / L, insulin 10 mg / L and indomethacin 100 mmol / L) Bone marrow mesenchymal stem cells differentiate into cardiomyocytes, vascular endothelial cells, osteoblasts and adipocytes. The cell morphology was also observed, and the differentiated cells were identified by immunocytochemistry and electron microscopy. Results The bone marrow mesenchymal stem cells could be isolated and cultured by gradient-adherent screening. 5-Azacytidine can induce stem cells to show myotubes, myofilaments and atrial granules, and myosin, Myosin, Zhuo-Actin, Troponin I immunostaining positive; VEGF can induce tubular network of bone marrow mesenchymal stem cells , Vascular-like and pebble-like structure, vWF immunostaining positive; osteoblast induction agent can induce differentiation of cells alkaline phosphatase positive; adipocyte induction agent differentiated cells appear fat droplets, oil red O staining shows lipid droplets orange red. Conclusion Adult canine bone marrow mesenchymal stem cells possess multi-directional differentiation potential in vitro. In the role of chemical or biological inducers, canine bone marrow mesenchymal stem cells can be differentiated into cardiomyocytes, vascular endothelial cells, osteoblasts, adipocytes and other mesenchymal tissue?