Tβ-4抑制梅花鹿鹿茸生长顶端间充质细胞软骨分化作用机制研究

Deer antler is the sole organ with periodic regeneration and an amazing growth rate in mammal. Studies showed that the rapid growth rate of deer antler depends on mesenchyme layer. In the layer, cell proliferation and differentiation is induced separately by different signal molecules. Therefore, a study on proliferation and differentiation of the cells in mesenchyme layer is beneficial to a better understanding of growth and ossification mechanism of deer antler. Currently, previous studies found many growth factors and the corresponding receptors related to antler growth, however, it is still unknown what factors play functions in maintaining the status of cell undifferentiation in mesenchyme layer during the so-long rapid growth period about 60 days. Our previous results revealed that thymosin beta 4 (Tβ-4) gene is an abundant expression gene in cDNA library of deer antler tip tissues. In addition, the gene expression is mainly focused on the mesenchyme layer; this implies that Tβ-4 gene may be a potential inhibitor of cellular cartilage differentiation in the mesenchyme layer. To date, related research reports are scarce, and the detailed molecular mechanisms are unknown. This project is aimed to probe the molecular mechanisms of Tβ-4 gene inhibiting cellular cartilage differentiation in the mesenchyme layer by using molecular biological technique and Digital Gene Expression Profiling (DGE); at the same time, it is expected to provide scientific basis for studying and curing bone diseases such as cartilage tissue defect on the basis of deer-antler based models.

鹿茸角是唯一具周期性再生能力的哺乳动物器官，且拥有惊人的生长速度。研究指出，鹿茸的快速生长取决于茸顶皮肤下的间充质层，该层细胞的增殖和分化分别由不同的信号分子诱导。研究鹿茸间充质细胞的增殖及分化，将为搞清鹿茸的生长与骨化机制提供依据。近年来，国际上对鹿茸角发育的研究普遍集中于细胞生长因子及其受体的发现上。鹿茸迅速发育时间大约为60天，这个过程中哪些细胞因子在维持间充质细胞长时间处于未分化状态呢？我们前期研究揭示thymosin beta 4 (Tβ-4)在鹿茸顶端cDNA文库中高丰度表达，且表达主要集中在间充质层，推测Tβ-4很可能为间充质细胞软骨分化潜在的抑制因子，但其详细分子机理不清，且无任何研究报道。本项目拟通过分子生物学和数字基因表达等技术以确定Tβ-4抑制间充质细胞软骨分化的分子调控机制，同时也为以鹿茸为哺乳动物模型开展软骨组织缺损修复等骨病的研究与治疗提供科学依据。

鹿茸是鹿体最活跃的生长点，间充质层为其干细胞储备层。鹿茸的变长是通过该层细胞经软骨内成骨过程实现的。我们的研究发现Tβ-4在cDNA 文库大规模ESTs测序中绝对高丰度表达，于是在本课题的基金资助下展开了对该种具有多重生物学功能的分子的研究：我们构建了Tβ-4-shRNA重组慢病毒表达载体，获得功能丧失的Tβ-4沉默突变体；构建了携带Tβ-4基因的过表达载体，并实现了细胞内Tβ-4蛋白稳定而高效表达；上调和抑制Tβ-4表达来研究该基因对间充质细胞生物学活性的影响，发现Tβ-4对间充质细胞软骨分化并没有影响，而是显著促进了细胞增殖；利用基于RNA-Seq的转录组测序技术研究Tβ-4影响的信号通路，KEGG分析显示，Tβ-4参与的信号通路主要包括PI3K/AKT、JNK/MAPK、NFκB、ILK/AKT等，GO分析结果指出，与Tβ-4沉默相关的差异mRNA 所对应的主要是与细胞增殖、细胞凋亡、血管发生、免疫调节等生理过程有关的基因。本研究揭示了Tβ-4对鹿茸间充质细胞的调控作用，并深入挖掘了Tβ-4介导的信号通路，为进一步深入研究Tβ-4在鹿茸发育中的分子机制及全面认识该基因功能奠定基础。