Runx对梅花鹿茸角软骨细胞分化的调控

发布时间：2018-02-26 06:10

  本文关键词： Runx 梅花鹿 鹿茸 软骨细胞 分化　出处：《吉林大学》2015年硕士论文　论文类型：学位论文

【摘要】：梅花鹿是我国的重要药用经济动物之一，其茸角作为一种传统的名贵中药而被广泛应用。鹿茸是一种独特的哺乳动物器官，其特点是可以完全再生，而其他哺乳动物器官均不具备在失去后完整再生的能力。因此，鹿茸角是研究哺乳动物器官再生的理想动物模型。Runx家族由3个基因编码区高度同源的成员Runx1、Runx2和Runx3组成，属于Runt结构域转录因子家族成员。大量研究发现，Runx在软骨细胞分化和成熟过程中起重要作用。Runx2在前肥大软骨细胞和肥大软骨细胞中高度表达，诱导软骨细胞的肥大化和软骨内骨化。进一步研究发现，Runx1和Runx3在软骨发育过程中也起到一定的调控作用，但有关Runx家族成员对梅花鹿茸角软骨细胞分化的调控却未见报道。 本实验以梅花鹿为研究对象，利用原位杂交、荧光定量PCR和RNA干扰方法对Runx1、Runx2和Runx3在梅花鹿茸角中的表达及其对鹿茸软骨细胞分化的影响进行了研究。原位杂交结果表明，Runx1、Runx2和Runx3高表达于梅花鹿鹿茸真皮层成纤维细胞、软骨膜纤维层、间充质层细胞及软骨细胞中，提示Runx1、Runx2和Runx3可能在梅花鹿茸角再生过程中起重要的作用。为了进一步研究Runx对鹿茸软骨细胞所起到的作用，在软骨细胞中添加Runx重组蛋白或转染Runx siRNA后，用荧光定量PCR方法检测软骨细胞标志分子（Col II、AGC和COMP）、肥大软骨细胞标志分子（Col X）和肥大前软骨细胞分泌因子IHH等表达的变化。结果发现，，经Runx1处理后的软骨细胞中，Col II、AGC、COMP、Col X和MMP13mRNA的表达水平均有一定程度的升高，而IHH mRNA在鹿茸软骨细胞中的表达则受到了明显的抑制；转染Runx1siRNA后，Col II、AGC、COMP、Col X和MMP13mRNA在鹿茸软骨细胞中的表达明显下降，而IHH的表达也有一定程度下降，提示Runx1具有抑制鹿茸软骨细胞向肥大前软骨细胞分化以及促进肥大前软骨细胞向肥大软骨细胞分化的作用。Runx2处理后的软骨细胞中，Col II、AGC和COMPmRNA的表达水平与对照组相比均有明显降低，而Col X、IHH和MMP13的表达量则有显著升高；转染Runx2siRNA后，鹿茸软骨细胞中Col II、AGC和COMPmRNA的表达水平均有显著升高，而Col X、和IHH的表达则明显降低，提示Runx2可能具有促进鹿茸软骨细胞成熟分化的作用。经Runx3处理后的软骨细胞中，ColII、AGC、COMP、Col X和MMP13mRNA的表达水平均有一定程度的下降，而IHH mRNA在鹿茸软骨细胞中的表达则有明显的升高；转染Runx3siRNA后，软骨细胞中Col II、AGC、COMP、Col X和MMP13mRNA表达量有显著升高，而IHH mRNA在鹿茸软骨细胞中的表达则受到了明显的抑制，提示Runx3可能具有促进软骨细胞向肥大前软骨细胞分化，并且抑制肥大前软骨细胞向肥大软骨细胞分化的作用。 综上所述，Runx1、Runx2和Runx3与鹿茸软骨细胞成熟分化过程密切相关。上述研究将为进一步探明梅花鹿鹿茸再生的调控机制提供理论基础。
[Abstract]:Sika deer is one of the important medicinal economic animals in China, and its antler horn is widely used as a traditional and valuable Chinese medicine. Therefore, antler antler is an ideal animal model for studying organ regeneration in mammals. Runx family consists of three highly homologous members, Runx1, Runx2 and Runx3. A member of the Runt domain transcription factor family. A large number of studies have found that Runx plays an important role in the differentiation and maturation of chondrocytes. Runx2 is highly expressed in premast chondrocytes and mast chondrocytes. Further studies show that Runx1 and Runx3 also play a role in the development of chondrocytes, but the regulation of Runx family on the differentiation of antler antler chondrocytes in sika deer has not been reported. In this experiment, sika deer was studied by in situ hybridization. Fluorescence quantitative PCR and RNA interference methods were used to study the expression of Runx1, Runx2 and Runx3 in antler antlers of sika deer and their effects on the differentiation of antler chondrocytes. The results of in situ hybridization showed that Runx1, Runx2 and Runx3 were highly expressed in Sika deer antler cortical fibroblasts. In chondrocyte fibrolayer, mesenchymal layer cells and chondrocytes, Runx1 Runx2 and Runx3 may play an important role in the regeneration of antler antlers of sika deer. In order to further study the effect of Runx on antler chondrocytes, Runx recombinant protein was added to chondrocytes or Runx siRNA was transfected into chondrocytes. Fluorescence quantitative PCR was used to detect the expression of chondrocyte markers (Col III-AGC and comp), mast chondrocyte marker (Col X) and IHH, the secretory factor of prehypertrophic chondrocytes. In chondrocytes treated with Runx1, the expression levels of Col 鈪

本文编号：1536815