本文關(guān)鍵詞： Runx 梅花鹿 鹿茸 軟骨細(xì)胞 分化　出處：《吉林大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：梅花鹿是我國的重要藥用經(jīng)濟(jì)動物之一，其茸角作為一種傳統(tǒng)的名貴中藥而被廣泛應(yīng)用。鹿茸是一種獨(dú)特的哺乳動物器官，其特點(diǎn)是可以完全再生，而其他哺乳動物器官均不具備在失去后完整再生的能力。因此，鹿茸角是研究哺乳動物器官再生的理想動物模型。Runx家族由3個基因編碼區(qū)高度同源的成員Runx1、Runx2和Runx3組成，屬于Runt結(jié)構(gòu)域轉(zhuǎn)錄因子家族成員。大量研究發(fā)現(xiàn)，Runx在軟骨細(xì)胞分化和成熟過程中起重要作用。Runx2在前肥大軟骨細(xì)胞和肥大軟骨細(xì)胞中高度表達(dá)，誘導(dǎo)軟骨細(xì)胞的肥大化和軟骨內(nèi)骨化。進(jìn)一步研究發(fā)現(xiàn)，Runx1和Runx3在軟骨發(fā)育過程中也起到一定的調(diào)控作用，但有關(guān)Runx家族成員對梅花鹿茸角軟骨細(xì)胞分化的調(diào)控卻未見報道。 本實(shí)驗(yàn)以梅花鹿為研究對象，利用原位雜交、熒光定量PCR和RNA干擾方法對Runx1、Runx2和Runx3在梅花鹿茸角中的表達(dá)及其對鹿茸軟骨細(xì)胞分化的影響進(jìn)行了研究。原位雜交結(jié)果表明，Runx1、Runx2和Runx3高表達(dá)于梅花鹿鹿茸真皮層成纖維細(xì)胞、軟骨膜纖維層、間充質(zhì)層細(xì)胞及軟骨細(xì)胞中，提示Runx1、Runx2和Runx3可能在梅花鹿茸角再生過程中起重要的作用。為了進(jìn)一步研究Runx對鹿茸軟骨細(xì)胞所起到的作用，在軟骨細(xì)胞中添加Runx重組蛋白或轉(zhuǎn)染Runx siRNA后，用熒光定量PCR方法檢測軟骨細(xì)胞標(biāo)志分子（Col II、AGC和COMP）、肥大軟骨細(xì)胞標(biāo)志分子（Col X）和肥大前軟骨細(xì)胞分泌因子IHH等表達(dá)的變化。結(jié)果發(fā)現(xiàn)，，經(jīng)Runx1處理后的軟骨細(xì)胞中，Col II、AGC、COMP、Col X和MMP13mRNA的表達(dá)水平均有一定程度的升高，而IHH mRNA在鹿茸軟骨細(xì)胞中的表達(dá)則受到了明顯的抑制；轉(zhuǎn)染Runx1siRNA后，Col II、AGC、COMP、Col X和MMP13mRNA在鹿茸軟骨細(xì)胞中的表達(dá)明顯下降，而IHH的表達(dá)也有一定程度下降，提示Runx1具有抑制鹿茸軟骨細(xì)胞向肥大前軟骨細(xì)胞分化以及促進(jìn)肥大前軟骨細(xì)胞向肥大軟骨細(xì)胞分化的作用。Runx2處理后的軟骨細(xì)胞中，Col II、AGC和COMPmRNA的表達(dá)水平與對照組相比均有明顯降低，而Col X、IHH和MMP13的表達(dá)量則有顯著升高；轉(zhuǎn)染Runx2siRNA后，鹿茸軟骨細(xì)胞中Col II、AGC和COMPmRNA的表達(dá)水平均有顯著升高，而Col X、和IHH的表達(dá)則明顯降低，提示Runx2可能具有促進(jìn)鹿茸軟骨細(xì)胞成熟分化的作用。經(jīng)Runx3處理后的軟骨細(xì)胞中，ColII、AGC、COMP、Col X和MMP13mRNA的表達(dá)水平均有一定程度的下降，而IHH mRNA在鹿茸軟骨細(xì)胞中的表達(dá)則有明顯的升高；轉(zhuǎn)染Runx3siRNA后，軟骨細(xì)胞中Col II、AGC、COMP、Col X和MMP13mRNA表達(dá)量有顯著升高，而IHH mRNA在鹿茸軟骨細(xì)胞中的表達(dá)則受到了明顯的抑制，提示Runx3可能具有促進(jìn)軟骨細(xì)胞向肥大前軟骨細(xì)胞分化，并且抑制肥大前軟骨細(xì)胞向肥大軟骨細(xì)胞分化的作用。 綜上所述，Runx1、Runx2和Runx3與鹿茸軟骨細(xì)胞成熟分化過程密切相關(guān)。上述研究將為進(jìn)一步探明梅花鹿鹿茸再生的調(diào)控機(jī)制提供理論基礎(chǔ)。
[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

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