人骨髓间质干细胞体外扩增和向内皮细胞定向诱导分化的研究

Amplification of mesenchymal stemcells from human bone marrow and ori-entation to induce MSCs differentiating into endothelial cells in vitro

目的:体外分离、扩增成人骨髓间质干细胞(MSCs)和向内皮细胞(ECs)定向诱导分化,开辟心血管组织工程种子细胞的新来源。方法:采用Percoll(1073g/L)从正常成人骨髓中分离出MSCs,纯化和扩增后流式细胞仪鉴定其纯度;用血管内皮生长因子(VEGF)诱导MSCs向ECs分化,Ⅷ因子(vWF)免疫组化和透射电镜(TEM)鉴定细胞性质。结果:5·0×105个MSCs在体外扩增15代后,获得8·0×1012个MSCs,扩增了约1·6×107倍;MSCs在加入VEGF诱导培养大约14-21d,80%-90%的诱导细胞对Ⅷ因子相关抗原呈阳性反应;TEM可观察到胞浆内有Weible-palade小体,证实为ECs。结论:成人骨髓MSCs在体外具有定向诱导分化为ECs的潜能,这为心脏组织工程瓣体外构建,尤其是在小儿先天性心脏病组织工程研究中种子细胞的来源提供了可能性。

AIM： Our purpose was to induce MSCs differentiating into endothelial cells （EC） in vitro and to provide the seed cells for study of cardiovascular tissue - engineering. METHODS： MSCs were separated by gradient centrifugation on Percoll （density 1 073 g/L） from human bone marrow （HBM）, and incubated for purification and amplification in DMEM （low glucose） with 10% fetal bovine serum （FBS）. Then, the MSCs were incubated for orientation differentiated into EC in DMEM （high glucose） with 20% FBS, VEGF （10μg/L）, bFGF （5μg/L）, L- glutamine （2 mmol/L）, penicillin （1 × 10^5U/L） and streptomycin（ 100 mg/L） for about 14 - 21 days and their phenotypic characteristics were analyzed by flow cytometry. Afterwards, the differentiating cells were evaluated by histology and immunohistochemistry.RESULTS： The quantity of MSCs was increased from 5.0 × 10^5 sin the primary culture to 8.0× 10^12, or to increase to 1.6 × 10^7 times after 15 generations of incubation. The purity of MSCs was above 95% and 98% homogeneous at passages 2 and 3, respectively. About 80% - 90% of the differentiating cells from MSCs after 14-21 days were positively stained for Ⅷ factor （vWF） related antigen by immunohistochemistry assay, and Weible- palade corpuscle was also observed by transmission electron microscopy in the cytoplasm. CONCLUSION： MSCs from HBM have the capability of differentiation into ECs in vitro, which may be a potential source of seed cells for fabrication of tissue - engineering heart valve, particularly in children with congenital heart disease.