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  

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

作　　者：张波[1] 杨述华[1] 张宇坤[1] 孙志博[1] 夏天[1] 许伟华[1] 叶树楠[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院骨科,武汉430022

出　　处：《中华创伤骨科杂志》2011年第2期160-164,共5页Chinese Journal of Orthopaedic Trauma

基　　金：基金项目：国家自然科学基金（30800654）

摘　　要：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100ng／mLGDF-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hBMSCs3周后，Ⅱ型胶原和AggrecanmRNA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。Objective To explore the feasibility and effectiveness of the self-assembly cartilage tissue engineered with cbondrogenically 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 hypoxie （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 II 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 normoxic 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 11 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. Conclusions Hypoxia may promote differentiation of hBMSCs into chondrocytes. The ＂ seff-assembly＂ technology can make it possible that e

关 键 词：低氧 细胞 软骨细胞 骨髓细胞 

分 类 号：R6[医药卫生—外科学]