摘要
[目的]探讨生长分化因子5(GDF-5)在诱导成人骨髓间充质干细胞(hBMSCs)成软骨分化的同时,促进其肥大成熟的作用。[方法]体外分离培养hBMSCs,取第四代细胞实验。将细胞消化、悬浮,以5×106/ml的细胞密度进行高密度培养,分别用含0、500 ng/ml GDF-5的软骨诱导液(CM)诱导培养3、4周。免疫组化、甲苯胺蓝染色检测II型胶原、X型胶原和蛋白多糖的表达情况。荧光实时定量RT-PCR检测两组微团II型胶原、X型胶原和蛋白多糖mRNA的表达情况。[结果]实验组微团中的细胞体积较对照组稍大,细胞密度稍低。甲苯胺蓝和免疫组化染色结果显示各组微团均有Ⅱ型胶原、X型胶原和蛋白多糖的表达和分泌,且4周实验组的表达最为强烈。荧光实时定量RT-PCR检测示,干预后3周实验组II型胶原、蛋白多糖基因表达量明显高于3周对照组(P<0.05),但X型胶原基因表达与对照组相比无显著性差异(P>0.05);干预后4周实验组X型胶原基因表达高于其余各组(P<0.05),Ⅱ型胶原、蛋白多糖基因表达明显高于两对照组(P<0.05),但与3周实验组相比较,差异无统计学意义(P>0.05)。[结论]GDF-5在诱导细胞成软骨分化的同时,还可促进其成熟肥大。
[ Objective] To study the effect of GDF -5 on the maturation and hypertrophy of chondrocytes. [ Methods] hMSCs were isolated and cultured in vitro, and the cells from passage 4 were used in this study. On reaching confluence, cells were digested, suspended and then centrifuged at a density of 5×10^6/ml to form pellets. The pellets were cultured for 3 or 4 weeks with CM supplemented with 500 ng/ml GDF - 5 or not. The expression of type Ⅱ collagen, type X collagen and glycosami- noglycan （GAG） was detected by immunohistochemistry and toluidine blue staining, respectively. Real- Time RT- PCR was performed to detect the expression of mRNA of type Ⅱ collagen, type X collagen and aggrecan. ~ Results] The chondrocytes in experiment groups were larger in volumn than those in control groups while the cell density was lower. The results of the histologi- cal staining showed that type Ⅱ collagen, type X collagen and GAG were expressed in all the groups, and reached the highest level at 4 weeks in the experiment group. The results of real - time RT - PCR showed that the mRNA expression of type Ⅱ colla- gen and aggrecan in the 3 - week experiment group was significantly higher than that in control groups （ P 〈 0. 05 ） while the mR- NA expression of type X collagen showed no significant difference （ P 〉 0. 05） . The mRNA expression of type X collagen in the 4 - week experiment group was significantly higher than that in any other group （ P 〈 O. 05 ）, the mRNA expression of type Ⅱ col- lagen and aggrecan was higher than that in control groups （P 〈 0. 05 ） and had no significant difference compared with the 3 - week experiment group （P 〉0. 05） . [ Conclusion] GDF-5 at 500 ng/ml could enhance chondrocyte maturation and hyper- trophy while inducing the chondrogenic differentiation.
出处
《中国矫形外科杂志》
CAS
CSCD
北大核心
2012年第17期1611-1614,共4页
Orthopedic Journal of China
关键词
成人骨髓间充质干细胞
生长分化因子5
软骨分化
成熟肥大
adults mesenchymal stem cells, GDF- 5, chondrogenic differentiation, maturation and hypertrophy
