脐血间充质干细胞的分离培养及生物学特性

背景：脐血富含干细胞，刺激后进入细胞周期的速度以及自泌生长因子的能力均强于骨髓及外周血干细胞。 目的：建立一种分离纯化、培养扩增脐血间充质干细胞的方法，并观察体外培养脐血间充质干细胞的生物学特性。 方法：密度梯度离心，贴壁培养和消化时间控制相结合，分离纯化脐血间充质干细胞。观察细胞形态，绘制细胞生长曲线，应用流式细胞仪测定细胞周期，以免疫组织化学法(SP法)测定CD29，CD44，CD34。采用体积分数20%马血清诱导脐血间充质干细胞分化为脂肪细胞，以油红O染色鉴定；0.1 μmol/L地塞米松，10 mmol/L β-甘油磷酸钠和50 μmol/L抗坏血酸诱导脐血间充质干细胞分化为成骨细胞，以碱性磷酸酶染色鉴定。 结果与结论：分离获得了高纯度贴壁生长的间充质干细胞，间充质干细胞呈梭形，细胞传代稳定，在体外连续传代培养9代，未发生形态学改变，无衰老征象。第3代间充质干细胞体外可以分化为脂肪细胞和成骨细胞。提示采用淋巴细胞分离液密度梯度离心，贴壁培养和消化时间控制相结合，可以获得纯度较高的脐血间充质干细胞，脐血间充质干细胞体外增殖和传代能力强。

Isolation,culture and biological characteristics of umbilical cord blood mesenchymal stem cells

BACKGROUND:Studies have found that cord blood contains a large member of hematopoietic stem cells and abundant mesenchymal stem cells. OBJECTIVE:To establish an isolating, culturing and proliferating method for human umbilical cord blood mesenchymal stem cells (HUMSCs) and then to observe cell biological characteristics in vitro. METHODS:HUMSCs were isolated and purified by density gradient centrifugation and adhering to the culture plastic. After successive subculture and amplification, the morphology was observed under microscope and the growth curve was drawn. Cell cycle was determined by flow cytometry; The expression of CD29, CD44 and CD34 in HUMSCs were detected by immunohistochemistry (SP); HUMSCs were induced to differentiate into adipocytes in DMEM with 20% horse serum and identified by oil red O staining; HUMSCs were induced to differentiate into osteoblasts by 0.1 μmol/L dexamethasone, 10 mmol/L β-glycerol phosphate sodium ascorbate and 50 μmol/L ascorbic acid and identified by alkaline phosphatase staining.   RESULTS AND CONCLUSION:High purity HUMSCs were isolated from umbilical cord blood. HUMSCs were spindle cells and their living behavior was quite stable. In vitro subcultured nine generations had no morphological changes and no signs of aging; Passage 3 of HUMSCs could differentiate into adipocytes and osteoblasts; The results showed that density gradient centrifugation, adherent culture and digestion time control can obtain an access to high purity HUMSCs with strong proliferation and passage ability in vitro.