Liao Xingen, Wu Lihua, Fu Mingfu, He Dingwen, Gu Yurong, Chen Weicai, Yin Ming
First Department of Orthopedics, the Second Affiliated Hospital of Nanchang University, Nanchang Jiangxi, 330006, P.R.China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2012 Jun;26(6):743-8.
To investigate the role of bone morphogenetic protein 2 (BMP-2) combined with hypoxic microenvironment in chondrogenic phenotype differentiation of bone marrow mesenchymal stem cells (BMSCs) of rat in vitro.
BMSCs were harvested from 4-week-old female Sprague Dawley rats. BMSCs at passage 2 were divided into 4 groups according different culture conditions: normoxia control group (group A), normoxia and BMP-2 group (group B), hypoxia control group (3% oxygen, group C), and hypoxia and BMP-2 group (group D). Then the cellular morphology was observed under inverted phase contrast microscope. Alcian blue immunohistochemical staining was used to detect the glycosaminoglycans (GAG), Western blot to detect collagen type II and hypoxia-inducible factor 1alpha (HIF-1alpha), and RT-PCR to detect the expressions of chondrogenic related genes, osteogenic related genes, and hypoxia related genes.
At 21 days after induction of BMP-2 and hypoxia (group D), BMSCs became round, cell density was significantly reduced, and lacuna-like cells were wrapped in cell matrix, while the changes were not observed in groups A, B, and C. Alcian blue staining in group D was significantly bluer than that in other groups, and staining became darker with induction time, and the cells were stained into pieces of deeply-stained blue at 21 days. Light staining was observed in the other groups at each time point. The expression level of collagen type II protein in group D was significantly higher than those in other groups (P < 0.05). HIF-1alpha protein expression levels of groups C and D were significantly higher than those of groups A and B (P < 0.05). The expressions of collagen II alpha1 (COL2 alpha1) and aggrecan mRNA (chondrogenic related genes) were highest in group D, while the expressions of COL1 alpha1, alkaline phosphatase, and runt-related transcription factor 2 mRNA (osteogenic related genes) were the highest in group B (P < 0.05). Compared with groups A and B, HIF-1alpha (hypoxic related genes) in groups C and D significantly increased (P < 0.05).
BMP-2 combined with hypoxia can induce differentiation of BMSCs into the chondrogenic phenotype, and inhibit osteoblast phenotype differentiation. HIF-1alpha is an important signaling molecule which is involved in the possible mechanism to promote chondrogenic differentiation process.
探讨骨形态发生蛋白2（BMP-2）联合低氧微环境在大鼠骨髓间充质干细胞（BMSCs）体外软骨形成表型分化中的作用。
从4周龄雌性Sprague Dawley大鼠中分离获取BMSCs。将第2代BMSCs根据不同培养条件分为4组：常氧对照组（A组）、常氧+BMP-2组（B组）、低氧对照组（3%氧气，C组）、低氧+BMP-2组（D组）。然后在倒置相差显微镜下观察细胞形态。采用阿尔辛蓝免疫组织化学染色检测糖胺聚糖（GAG），蛋白质印迹法检测Ⅱ型胶原蛋白和缺氧诱导因子1α（HIF-1α），逆转录聚合酶链反应（RT-PCR）检测软骨形成相关基因、成骨相关基因和缺氧相关基因的表达。
在BMP-2和低氧诱导21天后（D组），BMSCs变为圆形，细胞密度显著降低，细胞基质包裹着陷窝样细胞，而A、B、C组未观察到这些变化。D组的阿尔辛蓝染色明显比其他组更蓝，且随着诱导时间染色变深，在21天时细胞被染成深蓝色块状。其他组在各时间点染色均较浅。D组Ⅱ型胶原蛋白蛋白表达水平显著高于其他组（P<0.05）。C组和D组的HIF-1α蛋白表达水平显著高于A组和B组（P<0.05）。D组中Ⅱ型胶原α-链1（COL2α1）和聚集蛋白聚糖mRNA（软骨形成相关基因）的表达最高，而B组中COL1α1、碱性磷酸酶和 runt相关转录因子2 mRNA（成骨相关基因）的表达最高（P<0.05）。与A组和B组相比，C组和D组中的HIF-1α（缺氧相关基因）显著增加（P<0.05）。
BMP-2联合低氧可诱导BMSCs向软骨形成表型分化，并抑制成骨细胞表型分化。HIF-1α是参与促进软骨形成分化过程可能机制的重要信号分子。
