本文選題：睪酮 + 間充質(zhì)細(xì)胞��； 參考：《吉林大學(xué)》2017年碩士論文

【摘要】：鹿茸是哺乳動物唯一可以進(jìn)行完全再生的器官。隨著季節(jié)的交替變換,鹿茸角每年都會發(fā)生周期性的脫落和再生。鹿茸的快速生長過程涵蓋了多種組織,其細(xì)胞分裂增殖的速度遠(yuǎn)超過癌癥細(xì)胞,但此時的鹿茸細(xì)胞處于精確的調(diào)控下而不會發(fā)生癌變。因此,鹿茸的這種特性,使其成為研究創(chuàng)傷修復(fù)及哺乳動物器官再生的理想動物模型,是再生醫(yī)學(xué)研究的熱點(diǎn)。睪酮是雄性哺乳動物體內(nèi)最重要的雄激素,為骨骼生長及維持骨骼完整性所必須�，F(xiàn)已發(fā)現(xiàn),鹿茸的生長與體內(nèi)睪酮水平密切相關(guān),隨著鹿茸的季節(jié)性再生周期的循環(huán),雄鹿血清中睪酮含量會發(fā)生規(guī)律性的變化。有人認(rèn)為,睪酮具有誘導(dǎo)鹿茸頂端細(xì)胞增殖和分化的作用,高濃度的睪酮能夠刺激鹿茸細(xì)胞增殖,在發(fā)情期公鹿雄激素合成增加,這與此期骨化增加密切相關(guān)。青春前期將鹿去勢之后鹿茸不再發(fā)育。IHH是調(diào)節(jié)軟骨細(xì)胞增殖與分化的一個關(guān)鍵因子,與受體Ptc結(jié)合后,可解除Ptc對Smo的抑制作用,活化的Smo進(jìn)一步激活鋅指轉(zhuǎn)錄因子Gli,進(jìn)而調(diào)控下游靶基因的轉(zhuǎn)錄。Runx2是軟骨細(xì)胞分化過程中的一個轉(zhuǎn)錄因子,可誘導(dǎo)軟骨細(xì)胞分化成肥大軟骨細(xì)胞,加速軟骨細(xì)胞的成熟。但有關(guān)睪酮能否通過IHH信號通路及Runx2來影響鹿茸軟骨細(xì)胞的分化目前尚未見報道。本實(shí)驗以梅花鹿為研究對象,通過原位雜交方法對AR在梅花鹿茸角中的表達(dá)進(jìn)行研究;結(jié)果顯示AR m RNA在鹿茸間充質(zhì)細(xì)胞及軟骨細(xì)胞中均有表達(dá),說明雄激素及其受體在鹿茸生長過程中起重要作用。分離鹿茸間充質(zhì)細(xì)胞及軟骨細(xì)胞,添加一定濃度的睪酮,通過MTS和熒光定量PCR方法分別檢測睪酮對鹿茸間充質(zhì)細(xì)胞及軟骨細(xì)胞增殖與分化的影響。實(shí)驗結(jié)果表明,睪酮并不能明顯改變鹿茸間充質(zhì)細(xì)胞的增殖活性,但可促進(jìn)軟骨細(xì)胞標(biāo)志分子ColⅡ、AGC和COMP的表達(dá),提示睪酮可誘導(dǎo)鹿茸間充質(zhì)細(xì)胞向軟骨細(xì)胞分化。在鹿茸軟骨細(xì)胞中添加睪酮后,細(xì)胞的增殖活性顯著增強(qiáng),且肥大軟骨細(xì)胞標(biāo)志分子COL X m RNA的表達(dá)也明顯高于對照組。為了進(jìn)一步研究睪酮對鹿茸軟骨細(xì)胞分化的調(diào)控,我們檢測了睪酮對IHH信號通路相關(guān)因子表達(dá)的影響,結(jié)果發(fā)現(xiàn),睪酮可抑制IHH、Gli1在鹿茸軟骨細(xì)胞中的表達(dá),而促進(jìn)Gli3的表達(dá)。在鹿茸軟骨細(xì)胞中轉(zhuǎn)染IHH si RNA或添加IHH信號通路阻斷劑Cyc后再添加睪酮,COL X的表達(dá)顯著高于睪酮單獨(dú)處理組,轉(zhuǎn)染Gli1 si RNA后添加睪酮可導(dǎo)致COL X在鹿茸軟骨細(xì)胞中的表達(dá)顯著升高,而沉默Gli3則可減弱睪酮對COL X表達(dá)的調(diào)控。進(jìn)一步研究發(fā)現(xiàn),在鹿茸軟骨細(xì)胞中,睪酮可通過IHH來調(diào)控Gli1和Gli3的表達(dá)。與此同時,睪酮可促進(jìn)Runx2在鹿茸軟骨細(xì)胞中的表達(dá)。轉(zhuǎn)染Runx2 si RNA,睪酮對COL X表達(dá)的影響受到了明顯的抑制。進(jìn)一步的研究發(fā)現(xiàn),在鹿茸軟骨細(xì)胞中轉(zhuǎn)染IHH或Gli1 si RNA后再添加睪酮,Runx2的表達(dá)顯著高于睪酮處理組。IHH信號通路阻斷劑Cyc可增強(qiáng)睪酮對Runx2表達(dá)的調(diào)控。而與此相反,沉默Gli3可減弱睪酮對Runx2的誘導(dǎo)效應(yīng)。綜上所述,睪酮可誘導(dǎo)鹿茸間充質(zhì)細(xì)胞及軟骨細(xì)胞的增殖與分化,IHH信號通路和Runx2可介導(dǎo)睪酮對鹿茸軟骨細(xì)胞分化的調(diào)控,且睪酮可通過IHH信號通路來調(diào)節(jié)Runx2的表達(dá)。這些研究結(jié)果將為進(jìn)一步探明鹿茸角發(fā)育與再生的分子機(jī)理、鹿茸產(chǎn)量提高、哺乳動物器官再生及創(chuàng)傷修復(fù)動物模型建立、梅花鹿鹿茸資源的開發(fā)和利用等提供理論依據(jù)。
[Abstract]:Antler is the only organ in mammals that can be completely regenerated. With the alternation of seasons, the antler angle is periodically dropped and regenerated every year. The rapid growth of antlers covers a variety of tissues, and its cell division proliferates far more than cancer cells, but the antler cells are under precise control at this time. Therefore, the characteristics of antler, which makes it an ideal animal model for the study of trauma repair and the regeneration of mammalian organs, is a hot spot in the research of regenerative medicine. Testosterone is the most important androgen in male mammals, for bone growth and maintenance of bone integrity. The growth of antler and the body's testis have been found. The level of ketone is closely related. With the circulation of the seasonal regeneration cycle of the deer antler, the testosterone content in the serum of the deer will change regularly. It is believed that testosterone can induce the proliferation and differentiation of the apical cells of antler. High concentration of testosterone stimulates the proliferation of antler cells and increases the androgenic hormone synthesis in the estrus period, which is the same as that in this period. The increase of ossification is closely related..IHH is a key factor in regulating the proliferation and differentiation of chondrocytes after prepuberty, which is a key factor regulating the proliferation and differentiation of chondrocytes. After binding with receptor Ptc, it can relieve the inhibitory effect of Ptc on Smo. The activated Smo activates the zinc finger transcription factor Gli further, and then regulates the transcriptional.Runx2 of the downstream target gene to be the cartilage cell. A transcriptional factor in the process of differentiation can induce chondrocytes to differentiate into hypertrophic chondrocytes and accelerate the maturation of cartilage cells. However, there is no report on whether testosterone can affect the differentiation of antler cartilage cells through IHH signaling pathway and Runx2. This experiment was aimed at the sika deer as the research object and AR in plum blossom by in situ hybridization. The expression in the horn of antler was studied. The results showed that AR m RNA was expressed in the mesenchymal cells and chondrocytes of antler, indicating that androgen and its receptors play an important role in the growth of antler. The separation of antler mesenchymal cells and chondrocytes, testosterone with a certain concentration, and the detection of testosterone by MTS and fluorescence quantitative PCR, respectively. The experimental results show that testosterone does not significantly change the proliferation activity of the antler mesenchymal cells, but can promote the expression of Col II, AGC and COMP, suggesting that testosterone can induce the differentiation of the pilose antler mesenchymal cells to chondrocytes. After adding testosterone, the proliferation activity of the cells was significantly enhanced, and the expression of COL X m RNA was significantly higher than that of the control group. In order to further study the regulation of testosterone on the differentiation of antler cartilage cells, we detected the effect of testosterone on the expression of IHH signaling pathway related factors. The results showed that testosterone could inhibit IHH and Gli1 in IHH. The expression of Gli3 was promoted in the chondrocytes of pilose antler, and the expression of IHH Si RNA or IHH signaling pathway blocker Cyc was added to testosterone, and the expression of COL X was significantly higher than that of testosterone alone. The expression of testosterone after the transfection of Gli1 Si RNA could lead to a significant increase in the expression of COL cartilage in the deer cartilage cells. Li3 can weaken the regulation of testosterone on the expression of COL X. Further studies have found that testosterone can regulate the expression of Gli1 and Gli3 through IHH in antler cartilage cells. At the same time, testosterone can promote the expression of Runx2 in the antler cartilage cells. The effect of testosterone on the expression of Runx2 Si RNA is significantly inhibited. Further study of the effect of testosterone on COL X It was found that testosterone was added after transfection of IHH or Gli1 Si RNA in the antler cartilage cells. The expression of Runx2 was significantly higher than that in the testosterone treatment group,.IHH signaling pathway blocker Cyc enhanced the regulation of Runx2 expression by testosterone. On the contrary, the silence of Gli3 could weaken the induction effect of testosterone to Runx2. The proliferation and differentiation of chondrocytes, IHH signaling pathway and Runx2 mediate the regulation of testosterone on the differentiation of antler cartilage cells, and testosterone can regulate the expression of Runx2 through the IHH signaling pathway. These results will further explore the molecular mechanism of the development and regeneration of antler horn, the increase of the yield of antler, the regeneration of mammalian organs and the repair of trauma. The establishment of a complex animal model provides a theoretical basis for the development and utilization of sika deer antler resources.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S825

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 Jing Yang;Philipp Andre;Ling Ye;Ying-Zi Yang;;The Hedgehog signalling pathway in bone formation[J];International Journal of Oral Science;2015年02期

2 陳國芳;劉超;;睪丸功能與骨代謝關(guān)系的研究進(jìn)展[J];中華內(nèi)分泌代謝雜志;2015年04期

3 霍玉書;霍虹;張潔;;The Contribution of Deer Velvet Antler Research to the Modern Biological Medicine[J];Chinese Journal of Integrative Medicine;2014年10期

4 褚文輝;王大濤;魯曉萍;李春義;;基于干細(xì)胞的器官再生研究模型-鹿茸[J];中國組織工程研究;2013年45期

5 劉振;趙海平;楊春;褚文輝;王大濤;李春義;;鹿茸再生及其分子調(diào)節(jié)機(jī)理研究進(jìn)展[J];中國畜牧獸醫(yī);2013年02期

6 王欣;李光玉;李春義;;IGF1與睪酮激素對鹿角柄發(fā)生影響的研究[J];中國畜牧獸醫(yī);2010年10期

相關(guān)博士學(xué)位論文 前1條

1 李當(dāng)當(dāng);Hmgn在小鼠子宮蛻膜化過程中的作用及調(diào)節(jié)機(jī)制[D];吉林大學(xué);2016年

相關(guān)碩士學(xué)位論文 前1條

1 王守堂;PTHrP及其受體在梅花鹿茸角中的表達(dá)與調(diào)節(jié)[D];吉林大學(xué);2013年

，

本文編號：1909560