重组人骨形成蛋白2与骨诱导剂对大鼠骨髓间充质干细胞增殖分化的影响

目的观察重组人骨形成蛋白2(recombinant human bone morphogenetic protein2,rhBMP-2)与骨诱导剂对SD大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)的增殖与成骨作用。方法体外培养大鼠MSCs,分为实验组与对照组。对照组:不加rhBMP-2与骨诱导剂。实验组:骨诱导剂单独作用于SD大鼠MSCs(A组);rhBMP-2分别以浓度为10(B组)、50(C组)、100(D组)、200μg/L(E组)单独作用于SD大鼠MSCs;rhBMP-2分别以浓度为10(F组)、50(G组)、100(H组)、200μg/L(I组)联合骨诱导剂作用于SD大鼠MSCs。测定第3、6、9、12天的增殖状况(MTT法)、碱性磷酸酶(alkaline phosphatase,ALP)活性和骨钙素(osteocalcin,OC)水平。结果倒置相差显微镜观察SD大鼠MSCs原代培养时,细胞接种12h后即可贴壁;48h细胞成梭形,形似成纤维细胞;4d时细胞为多角形、纺锤形;6d时,成纤维细胞散在分布,少量呈集落样生长,为漩涡状、放射状排列;10d左右,细胞基本铺满瓶底,融合成片。传代细胞5~7d即可长满瓶底。各时间点A~I组均能显著促进MSCs成骨活性(ALP和OC)的表达。B~E组同时具有促进MSCs增殖作用,并呈浓度依赖性;F~I组增殖及ALP、OC含量均高于A~E组,比较差异有统计学意义(P<0.05)。结论rhBMP-2与骨诱导剂联合作用可在促进MSCs增殖的同时提高其成骨活性。

EFFECTS OF RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN 2 AND OSTEOGENIC AGENTS ON PROLIFERATION AND DIFFERENTIATION OF RAT MESENCHYMAL STEM CELLS

OBJECTIVE: To investigate the effects of the recombinant human bone morphogenetic protein 2 (rhBMP-2) and/or the osteogenic agents on proliferation and expression of the osteoblast phenotype differentiation of the SD rat mesenchymal stem cells (MSCs). METHODS: The rat MSCs were cultured in vitro and were randomly divided into the experimental groups (Groups A-I) and the control group. In the experimental group, MSCs were induced by rhBMP-2 in different doses (10, 50, 100 and 200 microg/L) in Groups B-E, the osteogenic agent alone (Group A) and by the combined use of rhBMP-2 in different doses (10,50, 100 and 200 microg/L)] and the osteogenic agent in Groups F-I. The MTT colorimetric assay was used to evaluate the proliferation, and the activities of alkaline phosphatase (ALP) and osteocalcin (OC) were observed at 3, 6, 9, 12 days, respectively. RESULTS: The inverted phase contrast microscopy showed that MSCs by primary culture for 12 hours were adhibited, with a fusiform shape at 48 hours. At 4 days they were polygonal or atractoid, and were spread gyrately or radiately at 6 days. At 10 days, they were spread at the bottom of the bottle. The statistical analysis showed that the expression of the osteoblast phenotype differentiation of MSCs could be induced in the experimental groups. The proliferation of MSCs could be enhanced in a dose-dependent manner in Groups B-E. The expression of the osteoblast phenotype differentiation, which was tested by the activities of ALP and OC, was significantly higher in Groups F-I than in Groups A-E. CONCLUSION: The combined use of rhBMP-2 and the osteogenic agents can enhance the MSC proliferation and induce an expression and maintenance of the osteoblast phenotype differentiation of the rat MSCs.