【摘要】 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?更多还原

【Abstract】 Objective To observe multilineage potential of adult canine mesenchymal stem cells, and provide experimental basis for restoration and stem cell transplantation of damaged tissue. Methods Mesenchymal stem cells were isolated and culture-expanded from canine bone marrow by gradient centrifugal and adhere to growth. Mesenchymal stem cells were differentiated committed into cardiomyocytes, endothelial cells, bone cells and adipose cells by means of 5-azacytidine(20 μmol/L), vascular endothelial growth factor(VEGF, 10 ng/ml),revulsant for bone cells(Dexamethasone 10nMol/L, Ascorbate 0.05mMol, β-Glycerol Phosphate, β-GP 10 mMol/L), and revulsant for adipose cells(1-methyl-3-isobutylxanthine 0.5mmol/L, Dexamethasone 1mol/L, Insulin 10mg/L, indomethacin 100mmol/L)respectively. The differentiation cells were identified by observing cellular configuration, immunohistochemical staining and electronic microscope technique. Results Mesenchymal stem cells could be isolated and culture-expanded from canine bone marrow by means of gradient centrifugal and adhere to growth method. Mesenchymal stem cells were induced to differentiate to myotubes, and appeared myofilaments and atrial granules in cytoplasm with 20 μmol/L 5-azacytidine. Myocyte antigens such as α-actin,α-actinin, myosin, and cardiomyocyte specific troponin I of differentiation cells were stained positively in immunohistochemistry. Mesenchymal stem cells were also induced to appear typical endothelial cell structure and vWF positive in immunohistochemistry with 10 ng/ml VEGF. Revulsant for bone cells could differentiate mesenchymal stem cells to bone cells, appearing an alkaline phosphatase positive in differentiation bone cells. Revulsant for adipose could differentiate mesenchymal stem cells to adipose cells, appearing adipose globelet and Oil Red O positive in cytoplasm. Conclusion Canine bone marrow mesenchymal stem cells could differentiate committed into cardiomyocytes, endothelial cells, bone cells and adipose cells by chemic and biological revulsants. So, adult canine mesenchymal stem cells have a multilineage potential in vitro.更多还原