睾酮对梅花鹿鹿茸间充质细胞及软骨细胞增殖与分化的影响

发布时间：2018-05-19 09:24

本文选题：睾酮 + 间充质细胞　；参考：《吉林大学》2017年硕士论文

【摘要】：鹿茸是哺乳动物唯一可以进行完全再生的器官。随着季节的交替变换,鹿茸角每年都会发生周期性的脱落和再生。鹿茸的快速生长过程涵盖了多种组织,其细胞分裂增殖的速度远超过癌症细胞,但此时的鹿茸细胞处于精确的调控下而不会发生癌变。因此,鹿茸的这种特性,使其成为研究创伤修复及哺乳动物器官再生的理想动物模型,是再生医学研究的热点。睾酮是雄性哺乳动物体内最重要的雄激素,为骨骼生长及维持骨骼完整性所必须。现已发现,鹿茸的生长与体内睾酮水平密切相关,随着鹿茸的季节性再生周期的循环,雄鹿血清中睾酮含量会发生规律性的变化。有人认为,睾酮具有诱导鹿茸顶端细胞增殖和分化的作用,高浓度的睾酮能够刺激鹿茸细胞增殖,在发情期公鹿雄激素合成增加,这与此期骨化增加密切相关。青春前期将鹿去势之后鹿茸不再发育。IHH是调节软骨细胞增殖与分化的一个关键因子,与受体Ptc结合后,可解除Ptc对Smo的抑制作用,活化的Smo进一步激活锌指转录因子Gli,进而调控下游靶基因的转录。Runx2是软骨细胞分化过程中的一个转录因子,可诱导软骨细胞分化成肥大软骨细胞,加速软骨细胞的成熟。但有关睾酮能否通过IHH信号通路及Runx2来影响鹿茸软骨细胞的分化目前尚未见报道。本实验以梅花鹿为研究对象,通过原位杂交方法对AR在梅花鹿茸角中的表达进行研究;结果显示AR m RNA在鹿茸间充质细胞及软骨细胞中均有表达,说明雄激素及其受体在鹿茸生长过程中起重要作用。分离鹿茸间充质细胞及软骨细胞,添加一定浓度的睾酮,通过MTS和荧光定量PCR方法分别检测睾酮对鹿茸间充质细胞及软骨细胞增殖与分化的影响。实验结果表明,睾酮并不能明显改变鹿茸间充质细胞的增殖活性,但可促进软骨细胞标志分子ColⅡ、AGC和COMP的表达,提示睾酮可诱导鹿茸间充质细胞向软骨细胞分化。在鹿茸软骨细胞中添加睾酮后,细胞的增殖活性显著增强,且肥大软骨细胞标志分子COL X m RNA的表达也明显高于对照组。为了进一步研究睾酮对鹿茸软骨细胞分化的调控,我们检测了睾酮对IHH信号通路相关因子表达的影响,结果发现,睾酮可抑制IHH、Gli1在鹿茸软骨细胞中的表达,而促进Gli3的表达。在鹿茸软骨细胞中转染IHH si RNA或添加IHH信号通路阻断剂Cyc后再添加睾酮,COL X的表达显著高于睾酮单独处理组,转染Gli1 si RNA后添加睾酮可导致COL X在鹿茸软骨细胞中的表达显著升高,而沉默Gli3则可减弱睾酮对COL X表达的调控。进一步研究发现,在鹿茸软骨细胞中,睾酮可通过IHH来调控Gli1和Gli3的表达。与此同时,睾酮可促进Runx2在鹿茸软骨细胞中的表达。转染Runx2 si RNA,睾酮对COL X表达的影响受到了明显的抑制。进一步的研究发现,在鹿茸软骨细胞中转染IHH或Gli1 si RNA后再添加睾酮,Runx2的表达显著高于睾酮处理组。IHH信号通路阻断剂Cyc可增强睾酮对Runx2表达的调控。而与此相反,沉默Gli3可减弱睾酮对Runx2的诱导效应。综上所述,睾酮可诱导鹿茸间充质细胞及软骨细胞的增殖与分化,IHH信号通路和Runx2可介导睾酮对鹿茸软骨细胞分化的调控,且睾酮可通过IHH信号通路来调节Runx2的表达。这些研究结果将为进一步探明鹿茸角发育与再生的分子机理、鹿茸产量提高、哺乳动物器官再生及创伤修复动物模型建立、梅花鹿鹿茸资源的开发和利用等提供理论依据。
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S825

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