TAP63γ 影响软骨内成骨发育的作用及机制研究

The protein p63 has been identified as a homolog of the tumor suppressor protein p53 and is consisted of many isoforms, including TAp63-α/β/γ and DeltaNP63-α/β/γ. Mice deficient of p63 showed limb malformations, including absent of limbs. Similar to p63-/- mice, p63 mutations can lead to congenital abnormal resulted in aplasia of limbs and/or ectoderm. These surveys prove that P63 plays an important role in skeletal development. Nevertheless, how these p63 isoforms impact long bone development is still a glimpse. Recently, in our research, increased TAp63γ expression was detected in hypertrophy chondrocytes, and furthermore, TAp63γ up-regulated Col10a1 expression and advanced hypertrophic differentiation of ATDC5 cells (an established cell model imitating endochondral ossification). Therefore, we infer that TAp63 promotes chondrocyte hypertrophic differentiation via upregulating Col10a1.To verify this, we intend to confirm the effect of TAp63γ on Col10a1 expression and chondrocyte hypertrophy by means of a series of cellular, molecular and biological methods. We also intend to launch TAp63γ gain- and loss-of-function studies in vivo to determine its effect and mechanisms on Col10a1 expression and endochondral ossification. The accomplish of the research will conspicuously help us to comprehend the mechanism of long bone development, prevention and treatment of related skeletal diseases likewise.

p63的异构体包括有TAp63-α、β、γ和△NP63-α、β、γ等。p63基因缺失小鼠表现为肢体发育畸形，其中包括四肢缺失。与p63基因缺失的表型相似，p63突变可导致以肢体和(或)外胚层发育不全为特征的先天异常。因此，p63被认为是骨骼发育所必需的。然而，人们对于p63的异构体如何影响骨骼发育仍是管中窥豹。最近，我们研究发现在肥大型软骨细胞成熟时TAp63γ高表达，且在ATDC5细胞（软骨内成骨模型）中，TAp63γ能上调Col10a1基因的表达，促进软骨细胞的分化和成熟。因此，我们推测TAp63可能通过调控Col10a1基因表达来影响软骨细胞的分化、成熟。为此，我们将通过体外细胞及TAp63γ功能获得性和缺失性研究，确定TAp63调控Col10a1基因表达与软骨细胞成熟的联系，探究其如何影响软骨内成骨发育。

p63基因是p53基因家族成员之一,在结构及功能上与p53具有一定的同源性,人类P63基因家族成员与机体的发育与肿瘤发生密切相关，因而受到了广泛关注。有研究显示，p63缺失会导致明显的发育异常，包括严重的皮肤、心脏和生殖系缺陷。除此以外，p63的缺失也会导致小鼠严重的上皮和肢体缺损，这表明在长骨发育过程中，p63也是软骨内成骨所必需的。根据启动子及选择性剪切的不同，p63可以编码多种亚型蛋白,发挥不同的生物学功能。在软骨内成骨的一个关键阶段——软骨细胞肥大中，p63其中一亚型TAp63γ水平升高。我们为了研究TAp63γ在软骨细胞分化和成熟中的作用，制备了稳定表达TAp63γ的ATDC5细胞系。检测发现与对照细胞相比，TAp63γ细胞在培养4天和7天后表现出明显的Col10a1表达上调。此外，TAp63γ细胞中的碱性磷酸酶、茜素红和阿尔新蓝染色更强，这些结果表明TAp63γ可以促进软骨细胞增殖、肥大分化和基质矿化。后期为了在体内研究TAp63γ在软骨内成骨过程中的作用，我们又建立了表达由Col10a1调控元件驱动的带flag标记的TAp63γ转基因小鼠。转基因小鼠出生后第1天的骨骼染色显示，与野生型同胞相比，长骨、尾骨和手指骨的骨化加速。此外，转基因小鼠长骨增殖和/或肥大区的Sox9表达减少且RUNX2表达增加。因此，我们认为这些结果表明TAp63γ促进软骨内成骨和骨骼的发育，至少可以部分通过控制软骨细胞的分化和成熟来完成。