人骨髓基质细胞体外分离及定向培养内皮细胞

Orientational Culture and Evaluation of Endothelial Cells from Human Bone Marrow Stromal Cells in Vitro

用Ficoll(比重1.077 g/ml)从正常成人骨髓中分离骨髓基质细胞(BMSCs),DMEM-HG 培养基内含20%FBS、GM-CSF(100 u/ml)、VEGF(10 ng/ml)、FGF(5 ng/ml)、L-谷氨酰胺(2mmol/ L)、肝素(90 u/ml),以及抗生素液进行定向培养和扩增其中的内皮细胞(ECs),Ⅷ因子相关抗原的免疫组化法和透射电镜观察(TEM)鉴定其细胞的性质。结果5.0×105个BMSCs在体外经定向ECs 培养和扩增8代后,获得了6.0×109个ECs,扩增了约1.2×104倍。70%-80%的细胞对Ⅷ因子相关抗原免疫组化呈阳性反应;光镜下细胞呈典型的“鹅卵石”样;TEM下可观察到胞浆内有Weible- palade小体,证实为内皮细胞。实验表明,BMSCs在体外分离和定向培养的ECs,经扩增后可能是心血管组织工程所需种子细胞的又一个重要来源。

Human bone marrow stromal cells (BMSCs) were separated by gradient centrifugation on Ficoll (density 1.077 g/ml) from human bone marrow (HBM) in vitro, orientational cultured and amplified endothelial cells (ECs) in DMEM (high glucose) with 20% fetal bovine serum (FBS), GM-CSF (100 u/ml), VEGF(10 ng/ml), FGF(5 ng/ml), L-glutamine (2 mmol/L), hepalean (90 u/ml) and antibiotics for about 21-28 days. The property of these ECs was evaluated by morphology with light microscopy and transmission electron microscopy (TEM), and immu-nohistochemistry with factor Ⅷ related antigen. After orientational cultured for endothelial cells, 5.0×105 of BMSCs in the primary culture was increased to 6.0×109 of ECs, or to increase 1.2×104 times after 8 generations incubated. More than 70%-80% of the cells from BMSCs cultured after 21-28 days were positive stain for factor Ⅷ related antigen by immunohistochemistry assay. The typical 'cobblestone' of these cells presented was observed by light microscopy. The Weible-palade corpuscle, which form is typical morphology of ECs, was also observed by TEM in the cytoplasm. The results showed that BMSCs from HBM had the capability in orientational culture for ECs, which after amplification in vitro might be another source of the seed cells in the cardiovascular tissue-engineering.