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

CTGF/Hcs24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Dig-， 35s标记的核糖探针，通过原位杂交分析小鼠发育过程中CTGF基因的表达模式。CTGF基因在发育过程中有2波表达，其中第1波在部分动物E9左右出现，第2波从E13开始上升，主要依赖于软骨内成骨的开始。通过软骨内成骨在肥大软骨细胞中观察到CTGF基因的特异位点表达，而在血管内皮细胞中观察到CTGF基因在各个阶段的表达。在导致钙化骨骼异常的Cbfal缺陷小鼠中，CTGF从未在软骨内骨样本中表达。我们认为CTGF在发育过程中可能具有双重作用，一是在早期发育中未知的作用，另一种作用是作为软骨内成骨过程中肥大软骨细胞分化的必要因素。在接下来的研究中，我们建立了在小鼠XI型胶原启动子控制下过量产生CTGF的转基因（Tg）小鼠。Tg小鼠能正常发育和生长。但他们在出生几个月后就出现了侏儒症。x线分析显示，与正常小鼠相比，骨密度降低。尤其是后肢的股骨密度明显较低。这些结果表明，CTGF的过表达影响软骨内成骨的某些步骤。此外，Tg小鼠的睾丸比正常小鼠小得多，生育能力受到影响，表明CTGF/Hcs24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.