FGF-2对人骨髓间充质干细胞增殖和向成骨细胞分化的影响

探讨体外培养条件下，成纤维细胞生长因子-2（FGF-2）和地塞米松（Dex）对第7代人骨髓间充质干细胞（MSCs）增殖和向成骨细胞分化的作用以及两者联合使用的效应。MSCs经含FGF-2或／和Dex的培养液作用后，于不同时间采用MTT法测定细胞增殖情况；对硝基苯磷酸（pNPP）法测定碱性磷酸酶（ALP）活性；ELISA法测定骨钙蛋白（OC）含量；茜素红S染色法对沉积的钙盐进行染色。发现：（1）FGF-2组细胞的生长速度为对照组的1．31倍，Dex／FGF-2组细胞的生长速度为FGF-2组的1．12倍。（2）Dex组的ALP活性、OC含量和细胞外基质钙盐沉积分别为对照组的17．0倍、2．12倍和10．56倍，并能形成成熟的羟基磷灰石（HA）结晶和骨结节；FGF-2组的ALP活性比对照组降低了76．7％，虽然OC含量、钙盐沉积增加，但不能形成成熟的HA结晶和骨结节；FGF-2对Dex诱导的ALP活性增加和HA结晶形成有拮抗作用。由此证明：（1）FGF-2可促进MSCs的增殖，Dex对MSCs的增殖无明显作用；Dex能增强FGF-2对MSCs的促增殖效应。（2）Dex可使MSCs分化为成熟的成骨细胞，是一个有效的成骨细胞分化诱导剂；FGF-2可使MSCs分化为未成熟的成骨细胞；FGF-2拮抗Dex诱导MSCs分化为成熟的成骨细胞。

Effects of FGF-2 on the Proliferation and Osteogenic Differentiation of the Adult Mesenchymal Stem Cells from Human Bone Marrow

To study the effects of fibroblast growth factor-2 (FGF-2) and/or dexamethasone (Dex) on the proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells (MSCs) from passage 7 (P7) in vitro. Following the treatment with different mediums containing FGF-2 and/or Dex in vitro, the proliferationof human bone marrow MSCs (from P7) was evaluated via MTT assay; for osteogenic differentiation, the alkaline phosphatase (ALP) activity was determined by biochemical colorimetric assay with pNPP, and the contents of osteocalcin (OC) was detected by ELISA assay at different times, and then the assay of extracellular matrix miner- alization was based on the detection of calcium mineral deposition using alizarin red S staining. When MSCs of P7 were cultured at low-cell-density in vitro, the growth rate of MSCs was 1.31-fold higher in the cultures treated with FGF-2 as compared to that of control at confluence. The growth rate of MSCs in the cultures treated with Dex/ FGF-2 increased 1.47-fold or 1.12-fold respectively compared to that of control or FGF-2 treated cultures. The growth rate of MSCs did not alter obviously in the cultures treated with Dex alone. Under osteogenic differentiation culture conditions, the treatment with Dex increased the ALP activity (17.0-fold), and OC contents (2.12-fold) of MSCs. Alizarin red S staining of cells indicated that Dex enhanced calcium mineral deposition in extracellular matrix (10.56-fold) and could mature mineral deposition into hydroxyapatite (HA) crystal and bone-like nodules. FGF-2 treatment decreased ALP activity (76.7%), increased OC contents and calcium mineral deposition of MSCs. In the treatment of FGF-2 alone, the MSCs formed amorphous calcium mineral deposition and failed to mature into HA crystal and bone-like nodules. Thus, FGF-2 antagonized the induction of Dex on the ALP and HA crystal formation of MSCs. Results suggest that FGF-2 increases the proliferation potential of MSCs while Dex alone has not this effect. The combination of FGF-2 and Dex increases the proliferation potential at a level much higher than the FGF- 2 alone. Dex can introduce MSCs into mature osteoblast as a potent osteogenic inducer. FGF-2 can also stimulates the osteogenic differentiation of MSCs, but the differentiated cells remain in immature state. FGF-2 antagonists the effect of inducing mature osteocytes by Dex.