Duan Zhixia, Zheng Qixin, Guo Xiaodong
Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan Hubei 430022, PR China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2007 Oct;21(10):1118-22.
To investigate the effect of the synthetic bone morphogenetic protein 2 (BMP-2)-derived peptide on the osteogenic induction in the marrow mesenchymal stem cells (MSCs) and to evaluate the osteoinductivity and dose-dependence of the BMP-2-derived peptide in vitro.
MSCs of 4-week old Wistar rats were separated and cultured. In the 3rd passage, the conditional culture medium was changed, in which the BMP-2-derived peptide in the following doses was added: 300,200, 100, 50, and 0 microg/ml, respectively (Groups A-E). The activity of alkaline phosphatase (ALP)and the amount of calcium deposition were meassured at 5, 10, 15 and 20 days during the culture with the conditional culture medium. The real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) was performed to measure the mRNA expressions of collagen type I, osteopontin (OPN), and osteocalcin (OCN) and to measure the osteoinductivity of the BMP-2-derived peptide in the different concentrations.
Under the inverted phase contrast microscope, MSCs cultured in the conditional culture medium for 3-4 days were changed in shape, from long fusiform to short fusiform or polygon. As the concentration of the BMP-2-derived peptide increased, the time for MSCs to change into the osteoblasts decreased. There was a significantly greater level of the ALP activity and amount of the calcium deposition in Groups A and B than in the other groups (P < 0.05). However, there was no significant difference between Group A and Group B (P > 0.05). The result of FQ-PCR showed that after MSCs were cultured in the different doses of the conditional culture medium for 14 days, the mRNA expressions of collagen type I, OPN and OCN were at higher levels. An increasing order in the level of the cycle threshold (Ct) was found in the following groups: A > B > C > D). Almost no expression was found in Group E. The Ct levels were significantly greater in Groups A and B than in Groups C and D (P < 0. 05). However, there was no significant difference between Group A and Group B (P > 0.05).
The BMP-2-derived peptide can greatly promote differentiation of MSCs into the osteoblasts, the promotion of osteogenesis has a dose-dependent pattern, and the best inducing dosage is 200 microg/ml.
研究合成骨形态发生蛋白2（BMP-2）衍生肽对骨髓间充质干细胞（MSCs）成骨诱导的影响，并在体外评估BMP-2衍生肽的骨诱导活性及剂量依赖性。
分离培养4周龄Wistar大鼠的MSCs。在第3代时更换条件培养基，分别添加以下剂量的BMP-2衍生肽：300、200、100、50和0μg/ml（A-E组）。在用条件培养基培养的第5、10、15和20天，测定碱性磷酸酶（ALP）活性和钙沉积量。进行实时荧光定量聚合酶链反应（FQ-PCR）以检测Ⅰ型胶原、骨桥蛋白（OPN）和骨钙素（OCN）的mRNA表达，并评估不同浓度BMP-2衍生肽的骨诱导活性。
在倒置相差显微镜下，在条件培养基中培养3-4天的MSCs形态发生改变，从长梭形变为短梭形或多边形。随着BMP-2衍生肽浓度的增加，MSCs转变为成骨细胞的时间缩短。A组和B组的ALP活性水平和钙沉积量显著高于其他组（P<0.05）。然而，A组和B组之间无显著差异（P>0.05）。FQ-PCR结果显示，MSCs在不同剂量的条件培养基中培养14天后，Ⅰ型胶原、OPN和OCN的mRNA表达水平较高。各实验组循环阈值（Ct）水平由高到低依次为：A>B>C>D）。E组几乎无表达。A组和B组的Ct水平显著高于C组和D组（P<0.05）。然而，A组和B组之间无显著差异（P>0.05）。
BMP-2衍生肽可显著促进MSCs向成骨细胞分化，其促骨生成作用呈剂量依赖性，最佳诱导剂量为200μg/ml。
