Analysis on the gene expression of CTGF, an endochondral ossification factor, during mouse development and its application for a bone regeneration

CTGF/Hcs 24是一种富含半胱氨酸的新型多功能生长因子，属于CCN基因家族。我们制备了地高辛、35 S标记的核酸探针，用原位杂交技术分析了CTGF基因在小鼠发育过程中的表达模式。CTGF基因在胚胎发育过程中呈两个波型表达，第一波高峰出现在E9左右的体节中，第二波从E13开始上升，主要取决于软骨内骨化的开始。CTGF基因在软骨内成骨过程中的肥大软骨细胞中有特异性表达，而在血管内皮细胞中的表达则在各个阶段均有表达。在Cbfal缺陷小鼠的情况下，这导致了骨骼异常的钙化，CTGF从来没有在软骨内骨的原基中表达。结论：CTGF在发育过程中可能具有双重作用，一个是在早期发育中的未知作用，另一个作用是作为软骨内骨化过程中肥大软骨细胞分化的必要因子。Tg小鼠可正常发育生长。但他们在出生后几个月内就表现出侏儒症。X射线分析显示，与正常小鼠相比，它们的骨密度降低。特别是后肢的股骨显示出明显的低密度。这些结果表明CTGF的过度表达影响了软骨内骨化的某些步骤。此外，睾丸比正常小得多，生育力受到影响，在Tg小鼠，表明CTGF/Hcs 24也可能调节睾丸的胚胎发育。

CTGF/Hcs24 is a cystein-rich novel multifunctional growth factor which belongs to CCN gene family. We prepared Dig-, 35S-labeled riboprobes to analyze CTGF gene expression pattern during mouse development by in situ hybridization. CTGF gene was expressed as 2 waves in development, the first peek of the wave was exhibited around E9 in somites and the second wave ascended from E13 mainly depended on a start of the endochondral ossification. A site specific CTGF gene expression was observed in the hypertrophic chondrocytes through the endochondral ossification, whereas expression in the endothelial cells of blood vessels was observed in every stages. In the case of Cbfal deficient mice, which resulted a skeletal abnormality in the calcification, CTGF was never expressed in the anlage of endochondral bones. We concluded that CTGF has dual possible roles in development, one is an unknown role in early development, another role is as an essential factor of hypertrophic chondrocytes differentiation during endochondral ossification.In the next study, we established transgenic(Tg) mice that over produce CTGF under the control of mouse type XI collagen promoter. Tg mice could develop and grow normally. But they showed dwarfism within a few months after birth. X-ray analysis revealed that their bone density was decreased compared with normal mice. The femurs in the hind limbs in particular showed an apparent low density. These results indicated that over expression of CTGF affects certain steps of endochondral ossification. In addition, the testes were much smaller than normal and fertility was affected in Tg mice, indicating that CTGF/Hcs24 may also regulate the embryonic development of the testes.