Clancy Brian M, Johnson Joyce D, Lambert Andre Jean, Rezvankhah Saeid, Wong Anthony, Resmini Christine, Feldman Jeffrey L, Leppanen Scott, Pittman Debra D
Division of Musculoskeletal Sciences, Wyeth, 200 Cambridge Park Drive, Cambridge, MA 02140, USA.
Bone. 2003 Jul;33(1):46-63. doi: 10.1016/s8756-3282(03)00116-9.
Endochondral bone formation has been fairly well characterized from a morphological perspective and yet this process remains largely undefined at molecular and biochemical levels. In vitro and in vivo studies have shown that human bone morphogenetic protein-2 (hBMP-2) is an important developmental growth and differentiation factor, capable of inducing ectopic bone formation in vivo. This study evaluated several aspects of the osteogenic effect of hBMP-2 protein injected into quadriceps of female C57B1/6J SCID mice. Mice were euthanized 1, 2, 3, 4, 7, and 14 days postinjection and muscles were collected for several methods of analysis. Hematoxylin and eosin-stained sections of muscles injected with formulation buffer showed no evidence of osteogenesis. In contrast, sections of muscles injected with hBMP-2 showed evidence of endochondral bone formation that progressed to mineralized bone by day 14. In addition, radiographs of mice injected with hBMP-2 showed that much of the quadriceps muscle had undergone mineralization by day 14. Labeled mRNA solutions were prepared and hybridized to oligonucleotide arrays designed to monitor approximately 1300 murine, full-length genes. Changes in gene expression associated with hBMP-2 were determined from time-matched comparisons between buffer and hBMP-2 samples. A gene expression profile was created for 215 genes that showed greater than 4-fold changes at one or more of the indicated time points. One hundred twenty-two of these genes have previously been associated with bone or cartilage metabolism and showed significant increases in expression, e.g., aggrecan (Agc1), runt related transcription factor 2 (Runx2), bone Gla protein 1 (Bglap1), and procollagens type II (Col2a1) and X (Col10a1). In addition, there were 93 genes that have not been explicitly associated with bone or cartilage metabolism. Two of these genes, cytokine receptor-like factor-1 (Crlf1) and matrix metalloproteinase 23 (Mmp23), showed peak changes in gene expression of 15- and 40-fold on days 4 and 7, respectively. In situ hybridizations of muscle sections showed that Mmp23 and Crlf1 mRNAs were expressed in chondrocytes and osteoblasts, suggesting a role for both proteins in some aspect of cartilage or bone formation. In conclusion, oligonucleotide arrays enabled a broader view of endochondral bone formation than has been reported to date. An increased understanding of the roles played by these gene products will improve our understanding of skeletogenesis, fracture repair, and pathological conditions such as osteoporosis.
从形态学角度来看，软骨内成骨过程已得到较为充分的描述，然而在分子和生化水平上，这一过程仍很大程度上未被明确。体外和体内研究表明，人骨形态发生蛋白-2（hBMP-2）是一种重要的发育生长和分化因子，能够在体内诱导异位骨形成。本研究评估了将hBMP-2蛋白注射到雌性C57B1/6J SCID小鼠股四头肌中所产生的成骨效应的几个方面。在注射后1、2、3、4、7和14天对小鼠实施安乐死，并收集肌肉用于多种分析方法。注射配制缓冲液的肌肉苏木精和伊红染色切片未显示有成骨迹象。相比之下，注射hBMP-2的肌肉切片显示有软骨内成骨迹象，到第14天时进展为矿化骨。此外，注射hBMP-2的小鼠的X光片显示，到第14天时，大部分股四头肌已发生矿化。制备了标记的mRNA溶液，并与设计用于监测约1300个小鼠全长基因的寡核苷酸阵列杂交。通过缓冲液和hBMP-2样品之间的时间匹配比较，确定与hBMP-2相关的基因表达变化。为215个基因创建了基因表达谱，这些基因在一个或多个指定时间点显示出大于4倍的变化。其中122个基因先前已与骨或软骨代谢相关，且表达显著增加，例如聚集蛋白聚糖（Agc1）、 runt相关转录因子2（Runx2）、骨钙蛋白1（Bglap1）以及II型（Col2a1）和X型（Col10a1）前胶原。此外，有93个基因尚未明确与骨或软骨代谢相关。其中两个基因——细胞因子受体样因子-1（Crlf1）和基质金属蛋白酶23（Mmp23），分别在第4天和第7天显示出15倍和4倍的基因表达峰值变化。肌肉切片的原位杂交显示，Mmp23和Crlf1 mRNA在软骨细胞和成骨细胞中表达，表明这两种蛋白在软骨或骨形成的某些方面发挥作用。总之，寡核苷酸阵列使我们对软骨内成骨过程有了比迄今报道更广泛的认识。对这些基因产物所起作用的进一步了解将增进我们对骨骼发生、骨折修复以及骨质疏松等病理状况的认识。
