骨痂组织冰冻切片前胶原及转化生长因子－β1基因表达的原位定位

目的观察骨痂组织中前胶原、转化生长因子－β１（ｔｒａｎｓｆｏｒｍｉｎｇｇｒｏｗｔｈｆａｃｔｏｒβ１，ＴＧＦ－β１）基因表达模式，分析ＴＧＦ－β１在骨折愈合过程中的作用，探索骨组织冰冻切片原位杂交技术。方法采用不脱钙的大鼠骨痂组织冰冻切片进行原位杂交，观察骨痂组织中前胶原和ＴＧＦ－β１基因的表达，并与先前研究作对照。结果杂交信号清晰，定位良好，特异性高。骨折第１周末，成纤维细胞的Ⅲ型前胶原基因表达占主导，Ⅰ型前胶原ｍＲＮＡ阳性成骨细胞也出现于膜内化骨区。ＴＧＦ－β１在分化、增殖的成骨细胞以及接近成熟的软骨细胞有显著表达。骨折第２周末，Ⅱ型前胶原和ＴＧＦ－β１ｍＲＮＡ在成熟的软骨细胞大量表达，而Ⅰ型前胶原ｍＲＮＡ表达也明显增加。骨折第４周末，软骨骨痂基本被骨组织替代，见散在Ⅰ型前胶原ｍＲＮＡ表达阳性的成骨细胞。同时证实共有表型表达的现象存在。结论实验结果与以往有关研究结果的吻合，提示ＴＧＦ－β１在骨折愈合过程中，尤其在细胞分化、增殖中，起重要的调节作用。同时，也说明了此方法是一种快捷、灵敏、又不失特异性的骨组织原位杂交方法。

In Situ Localization of Procollagen and Transforming Growth Factor Gene Expression on Cryosection of Fracture Callus

ObjectiveTo investigate the expression mode of procollagen and transforming growth factor 1(TGF-1) gene in fracture callus, analyze the role of TGF-1 in fracture healing, and grope for the technique of in situ hybridization for skeletal tissue. MethodsIn situ localization of procollagen and TGF-1 gene expression was performed on the cryosection of rat fracture callus, and the results were compared with those of earlier study. ResultsThe hybridization signals were clear and easy to be localized with high specificity. On the seventh day after fracture, the expression of pro 1() in fibroblast and chondrocytelike cells was dominant. TGF-1 was strongly expressed in differentiating proliferating osteoblasts and premature chondrocytes. The end of second week was characterized by a marked increase in the mRNA levels of type-procollagen and TGF-1 in mature chondrocytes. At the end of fourth week, the cartilaginous callus was almost replaced by the osseous tissue. Some type-I procollagen mRNA positive osteoblasts and hypertrophic chondrocytes were found scattering in the woven bone and remains of cartilaginous callus. The levels of TGF-1 mRNA in positive cells were very low. ConclusionTGF-1 played an important role in fracture healing. The phenomenon of shared phenotype expression in developin tissues may provide an important approach to reveal the mechanism of the origin, differentiation, and orientation of cells. The method applied in this study was an easier, quicker, more sensitive and with high specificity.