低氧环境和生长分化因子-5联合诱导促进人骨髓基质干细胞分化成软骨细胞的研究

目的 探讨低氧环境下生长分化因子-5 (GDF-5)诱导人骨髓基质干细胞(hBMSCs)“自组装”形成工程化软骨的可行性和有效性。方法 分离培养hBMSCs，流式细胞学方法鉴定。将hBMSCs用含100 ng/mL GDF-5软骨诱导液(CM)分别在低氧（A组）和正常氧（B组）环境下诱导培养3周，RT-PCR检测Ⅱ型胶原和Aggrecan表达情况。将A、B两组hBMSCs消化后，按一定密度接种于2％琼脂糖包被的24孔板，在不同氧浓度下CM继续诱导3周，免疫组化法检测组织Ⅰ、Ⅱ型胶原表达，甲苯胺蓝染色检测葡萄糖胺聚糖(GAG)表达。结果 hBMSCs呈梭形漩涡状生长，高表达CD44、CD29，不表达CD45分子。诱导5d后A组细胞较B组明显增殖，诱导10dA组细胞体积较B组小。含GDF-5的CM诱导hBMSCs 3周后，Ⅱ型胶原和Aggrecan mRNA表达阳性，A组Ⅱ型胶原和Aggrecan表达量较B组均明显增加，差异有统计学意义(P＜0.05)。GDF-5诱导hBMSCs可“自组装”形成一定形状大小类软骨样组织，A组Ⅱ型胶原表达较B组增加，A组Ⅰ型胶原表达量下降，B组表达阳性；A组甲苯胺蓝染色异染加深。结论 低氧促进GDF-5诱导hBMSCs向软骨分化，低氧环境下GDF-5诱导软骨分化的hBMSCs“自组装”形成的工程化软骨更具有软骨表型。

Chondrogenic differentiation of human bone mesenchymal stem cells treated with growth differentiation factor 5 under hypoxia

ObjectiveTo explore the feasibility and effectiveness of the self-assembly cartilage tissue engineered with chondrogenically differentiated human bone mesenchymal stem cells (hBMSCs) induced by growth differentiation factor-5 (GDF-5) under hypoxia.Methods After hBMSCs were isolated from ilium, flow cytometry was performed to detect the cell surface markers, hBMSCs were exposed to cartilage inducing medium containing 100 ng/mL of GDF-5 in hypoxic (2％ oxygen) and normoxic (21％ oxygen)conditions, respectively. RT-PCR was performed to detect the expressions of type Ⅱ collagen and aggrecan in the cells after 3 weeks. In hypoxic and normoxic conditions, the hBMSCs were induced in conditioned medium (CM) containing GDF-5 for 3 weeks. After digestion, the hBMSCs, according to a certain density, were inoculated on 24-well plates coated with 2％ of agarose. Induction in the CM continued for another 3 weeks.Examples of both groups were detected for types Ⅰ and l collagen with histology and immunohistochemistry.Toluidine blue staining was used to detect the expressions of cartilage-specific components.Results The hBMSCs grew in a manner of spindle-shaped whirlpool, with high expressions of CD44 and CD29 and a negative expression of CD45. The hBMSCs exposed to the CM containing GDF-5. in the hypoxic condition, expressed type Ⅱ collagen and aggrecan significantly more than those exposed to the chondrogenic growth factors in the nonnoxic condition ( P ＜ 0. 05). The hBMSCs, in the hypoxic condition, after induction by GDF-5,inoculation on agar surface and induction in the CM, were self-assembled to form cartilage-like tissue in proper shape and size. In the hypoxia group, the expression of type Ⅱ collagen was increased and the expression of type Ⅰ collagen was decreased. The toluidine blue staining in the hypoxia group was deeper than in the normoxia group.ConclusionsHypoxia may promote differentiation of hBMSCs into chondrocytes.The ” self-assembly” technology can make it possible that chondrogenically differentiated hBMSCs induced by GDF-5 under hypoxia can be built in vitro into successful tissue-engineered cartilage.