INDUCTION OF RAPID OSTEOBLAST DIFFERENTIATION IN RAT BONE-MARROW STROMAL CELL-CULTURES BY DEXAMETHASONE AND BMP-2
INDUCTION OF RAPID OSTEOBLAST DIFFERENTIATION IN RAT BONE-MARROW STROMAL CELL-CULTURES BY DEXAMETHASONE AND BMP-2
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DOI：
10.1006/dbio.1994.1022
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发表时间：
1994-01-01
影响因子：
2.7
通讯作者：
KAZHDAN, I
中科院分区：
文献类型：
--
作者：
RICKARD, DJ;SULLIVAN, TA;KAZHDAN, I
成年脊椎动物在骨折修复过程中需要成骨细胞的持续供应来进行骨重建和再生。这意味着体内存在一个能够成骨的细胞库。这些骨祖细胞的一个来源是骨髓基质的成纤维细胞成分中的干细胞。成熟成骨细胞的特征在于高碱性磷酸酶和骨桥蛋白水平，结合骨特异性基质蛋白骨钙素和骨唾液蛋白的表达以及基质矿化的能力。我们已经使用这些标记物来定义允许从培养的骨髓基质细胞快速分化成骨细胞的条件。成骨细胞分化诱导连续培养与10- 5 M地塞米松（dex），刺激碱性磷酸酶（AP）的活性和mRNA水平，以及我们的骨桥蛋白，骨涎蛋白和骨钙素mRNA的培养第8天;共添加10- 5 M 1，25-二羟维生素D3（维生素D）与dex是必要的高骨钙素mRNA的表达。重组骨形态发生蛋白-2（BMP-2）与地塞米松具有相似的作用，并与地塞米松协同作用，大大提高了AP活性，增加了成骨细胞mRNA的水平。使用原位杂交检测单个细胞中mRNA的存在，结果表明，骨桥蛋白mRNA的出现先于AP mRNA，并且早在第4天就在dex处理的细胞集落中表达。通过流式细胞术定量细胞表面AP蛋白表明，与dex或BMP-2的培养物产生了低AP的细胞（暗淡细胞）和高AP水平的细胞的混合群体，而dex + BMP-2的组合产生了非常少的暗淡细胞和比单独使用任一诱导剂含有更高AP水平的细胞群体。当从dex处理的细胞中分选出暗淡的群体并用诱导剂再培养时，这些培养物产生高AP水平，并且能够存款矿化基质。因此，用诱导剂处理骨髓基质细胞产生成熟成骨细胞群体以及在进一步暴露于诱导剂时保留成骨细胞分化能力的未分化细胞群体。这些数据表明，骨髓基质隔室内的干细胞能够快速获得成骨细胞的功能，并建议在成骨发育阶段糖皮质激素与BMP-2和维生素D的组合的潜在作用。
Adult vertebrates require a continuous supply of osteoblasts for both bone remodeling and regeneration during fracture repair. This implies the existence of a reservoir of cells in the body capable of osteogenesis. One source of these osteoprogenitors is the stem cells within the fibroblastic component of bone marrow stroma. Mature osteoblasts are characterized by high alkaline phosphatase and osteopontin levels, combined with expression of the bone-specific matrix proteins osteocalcin and bone sialoprotein and the capacity for matrix mineralization. We have used these markers to define the conditions permitting rapid osteoblast differentiation from cultured bone marrow stromal cells. Osteoblastic differentiation was induced by continuous culture with 10-5Mdexamethasone (dex) which stimulated alkaline phosphatase (AP) activity and mRNA levels as well us osteopontin, bone sialoprotein, and osteocalcin mRNA by Day 8 of culture; coaddition of 10-5M1,25-dihydroxyvitamin D3(vitamin D) with dex was essential for high osteocalcin mRNA expression. Recombinant bone morphogenetic protein-2 (BMP-2) exerted similar effects to dex and acted in synergy with dex to yield greatly elevated AP activity as well as increased levels of osteoblastic mRNAs. Usingin situhybridization to detect the presence of mRNAs in individual cells, it was shown that appearance of osteopontin mRNA preceded AP mRNA, and was expressed in dex-treated cell colonies as early as Day 4. Quantitation of cell surface AP protein by flow cytometry indicated that culture with dex or BMP-2 produced a mixed population of cells with low AP (dim cells) and cells with high AP levels, while the combination of dex + BMP-2 yielded very few dim cells and a population of cells containing higher AP levels than with either inducer alone. When the dim population from dex-treated cells was sorted and recultured with inducers, these cultures developed high AP levels and were able to deposit a mineralized matrix. Thus, treatment of marrow stromal cells with inducer results in a population of mature osteoblasts as well as a population of undifferentiated cells which retains the capacity for osteoblastic differentiation with further exposure to inducers. These data demonstrate that stem cells within the stromal compartment of bone marrow are capable of rapidly acquiring osteoblast features and suggest a potential role for glucocorticoids in combination with BMP-2 and vitamin D in stages of osteogenic development.