骨形态发生蛋白4(BMP4)基因表达在骨折愈合过程中的定位研究

Demonstration and Localization of Bone Morphogenetic Protein 4(BMP4) in Fracture Healing

创伤后，骨组织的再生修复十分完全，其修复常无瘢痕残留。骨组织之所以有如此完美的愈合能力，是因为骨骼组织内存在有成骨因子，即骨生长因子。骨形态发生蛋白（ｂｏｎｅｍｏｒｐｈｏｇｅｎｅｔｉｃｐｒｏｔｅｉｎ，ＢＭＰ）因其直接诱导软组织成骨而受重视。本文应用ＢＭＰ４ｃＤＮＡ探针检测骨折愈合过程中外骨痂内ＢＭＰ４基因表达的定位与分布，探讨ＢＭＰ４基因表达在闭合性骨折愈合外骨痂形成中的作用。用６４只健康ＳＤ大鼠制备闭合性胫骨骨折动物模型。分别于骨折后１２小时、１、３、５、７、９、１４及２８天取材。取材后行恒冷切片，用地高辛素标记的ＢＭＰ４ｃＤＮＡ探针进行原位核酸分子杂交。大鼠骨折后１２小时～３天，骨折周围血肿内细胞及肌肉中新出现的间充质细胞内ＢＭＰ４ｍＲＮＡ表达检测为阳性信号。表明创伤激活ＢＭＰ４ｍＲＮＡ的表达，并呈区域性参与骨折的修复，也说明骨折血肿及周围软组织在骨折愈合过程中具有非常重要的地位。

The process of fracture healing by bone regeneration is a perfect and complete cascade without scarring.The growth factors found in bone are responsible for the wonderful healing.The bone morphogenetic proteins (BMPs) have been widely investigated due to its capacity to induce new bone formation in soft tissues.We used bone morphogenetic protein 4 cDNA probe to investigate the expression and localization of BMP4 mRNA in order to get a further understanding of closed fracture healing.Sixty four Sprague Dawley rats were used.Closed fractures were produced manually in the middle of the shaft of tibia after intramedullary fixation.Eight animals were killed each at 12 hours,1,3,5,7,9,14,and 28 days after operation.The external calluses were dissected with the surrounding tissue.The specimens were cut with use of a cryostat into 7 μm serial sections.The slides were in situ hybridized with Dig-11-dUTP labeled BMP4 cDNA probe and counterstained with Saffranin O.The control groups were included.Typical histologic features of endochondral ossification in external callus were seen in this experimental model.The results of in situ hybridization revealed that from 12 to 72 hours after fracture,the signals of BMP4 mRNA were positively detected and localized in the cells in parafracture hematoma and in the newly appeared mesenchymal cells in the muscles adjacent to the fracture site (DOPC and IOPC).The cells in parafracture hematoma might be from bone marrow.The control groups showed negative detection.The present results suggest that the trauma may trigger off the BMP4 gene expression in a specific callus forming regions as parafracture soft tissue before callus formation.The expression of BMP4 mRNA is a very important contributing factor in callus formation during the period of fracture healing,and in new bone formation induced by fracture hematoma which had osteogenetic potential.