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目的 确定人间充质干细胞 (MSCS)体外扩增培养、向成骨细胞表型转化的方法 ,了解其生物学特性变化。方法 分离人骨髓细胞 ,按不同种植密度、首次换液时间进行原代培养 ,对传代培养细胞用化学药物或生长因子进行成骨诱导培养 ,并检测成骨细胞表型、生长曲线、贴壁率。结果 人MSCS 原代培养种植细胞密度为 1× 10 5～ 3× 10 5/cm2 ,48h后半量换液 ,以后每 3d全量换液的方法较合理。经化学药物 (如地塞米松、β 甘油磷酸、维生素C)或生长因子 (rh BMP2 、BMPS)作用后 ,MSCS 表达碱性磷酸酶、Ⅰ型胶原、Ⅲ型胶原和骨钙素 ,细胞增殖速度减慢 ,贴壁率略降低。未诱导MSCS 形成细胞团后 ,可自动分化表达碱性磷酸酶活性。结论 合理的人MSCS 培养方法及可靠的诱导条件 ,可为体外构建组织工程化骨提供自体种子细胞。
Objective To determine the method of in vitro expansion of human mesenchymal stem cells (MSCs) to transform into osteoblast phenotype and to understand the changes of their biological characteristics. Methods Human bone marrow cells were isolated and cultured at different planting densities for the first time. The cultured cells were subcultured with chemical drugs or growth factors, and osteoblast phenotype, growth curve, adherent rate . Results The density of MSCS cultured in primary culture was 1 × 10 5 ~ 3 × 10 5 / cm 2. After 48 h, half of the liquid was replaced, and then the method of whole liquid exchange every 3d was reasonable. MSCs expressed alkaline phosphatase, type I collagen, type III collagen and osteocalcin after being treated with chemical drugs (such as dexamethasone, betaglycerophosphate, vitamin C) or rh BMP2 (BMPS) Slow down, adherent rate slightly lower. After not inducing MSCS forming cell mass, it can differentiate and express alkaline phosphatase activity automatically. Conclusion The reasonable MSCS culture method and reliable induction conditions can provide autologous seed cells for the construction of tissue-engineered bone in vitro.