【作者】 陈秀霞；

【导师】 侯加法；

【作者基本信息】 南京农业大学 ， 临床兽医学， 2009， 博士

【摘要】 雌激素(Estrogen)和雄激素(Androgen)对动物正常骨骼生长和代谢有着重要的调节作用。蛋鸡产蛋前形成大量髓质骨,为产蛋期蛋壳的形成提供钙源。雌激素和雄激素与髓质骨的产生密切相关,激素主要通过受体途径参与成骨细胞功能的调节,影响成骨细胞的增殖、凋亡、细胞周期及骨代谢相关基因的表达。鉴于雌激素和雄激素在髓质骨形成过程中的重要调节作用,本文通过雌激素和雄激素单独及联合应用研究其对髓质骨形成的影响,并对体外培养成骨细胞功能的变化进行研究,进一步探讨髓质骨形成的机理。试验Ⅰ、雌、雄激素对笼养青年蛋鸡骨代谢的影响目的：研究雌、雄激素对青年ISA蛋鸡骨代谢的影响。方法：选择300只92日龄ISA蛋鸡,随机平均分为5组(A组为空白对照组、B组为溶剂对照组、C组为雌激素组、D组为雄激素组、E组为雌、雄激素联合应用组),试验为期20天。每5天采集血样测定血钙(Ca)、血磷(P)、碱性磷酸酶(AKP)、抗酒石酸酸性磷酸酶(StrACP)水平,处死后测定骨质量及皮质骨形态。结果：与空白对照组乖溶剂对照组相比,从试验第10天开始,雌激素组肱骨质量及血浆AKP水平显著升高(P<0.05),雄激素组肱骨质量、胫骨质量、皮质骨厚度、皮质骨面积、皮质骨面积比例以及AKP水平显著升高(P<0.05)；雌、雄激素联合应用组肱骨质量、皮质骨面积比例、血钙以及AKP水平显著升高(P<0.05)。从试验第15天开始,雌激素组Ca、P水平显著升高(P<0.05),而StrACP显著降低(P<0.05)；雌、雄激素联合应用组皮质骨厚度显著增加(P<0.05)；试验第20天,雌、雄激素联合应用组皮质骨内径减小而外径增大。空白对照组和溶剂对照组之间无显著差异(P>0.05)。小结：雌激素能促进青年期蛋鸡骨形成,抑制骨吸收；雄激素能促进青年期蛋鸡皮质骨发育和成熟；雌、雄激素联合应用能促进骨生成和骨吸收,使骨转化维持在一个较高水平,促进骨骼发育。试验Ⅱ、雌、雄激素对髓质骨形成的影响目的：研究雌激素雄激素单独以及联合应用对髓质骨形成的影响。方法：选择300只92日龄ISA蛋鸡和60只92日龄阉割的ISA公鸡预饲1周后试验。A组为空白对照组、B组为溶剂对照组、C组为雌激素组、D组为雄激素组、E组为雌、雄激素联合应用组,F组为雌、雄激素联合应用阉割公鸡组,试验为期20 d。每5天杀鸡采集骨组织用于骨切片制作,甲苯胺蓝染色；测定血浆雌二醇(E2)和睾酮(T)水平。结果：A、B、D组未见或只有少量髓质骨产生；C组在试验第5天开始产生髓质骨,E组和F组均在试验第10天开始大量出现髓质骨。与A、B组比较,试验第5天,C组和E组组血浆雌二醇水平显著升高(P<0.05),而睾酮水平显著降低(P<0.05)；D组雌二醇水平显著降低(P<0.05),而睾酮水平升高(P<0.05)；F组雌二醇水平显著升高(P<0.05)而睾酮水平显著降低(P<0.05)。小结：髓质骨的产生与体内雌二醇和睾酮水平有关,雄激素单独作用不能诱导髓质骨的产生,雌激素单独以及雌、雄激素联合应用可诱导青年期ISA蛋鸡髓质骨的生成。试验Ⅲ、雌、雄激素对蛋鸡骨组织内ER、AR蛋白及mRNA表达水平的影响目的：研究雌激素受体(ER)和雄激素受体(AR)在骨组织中的定位,进一步研究外源性雌、雄激素对其受体作用及对其,mRNA表达水平的影响。方法：应用免疫组织化学法对骨组织内ER、AR蛋白进行定位,并对阳性表达率进行分析,通过半定量RT-PCR检测受体基因的相对表达。结果：免疫组化结果显示ER分布于成骨细胞和破骨细胞的细胞核和细胞浆中,雌激素及雌、雄激素联合应用可提高ER的阳性细胞表达率；AR在软骨细胞细胞核及细胞浆内均有表达,但在髓腔内未见阳性细胞。半定量RT-PCR结果表明雌激素可以诱导ER mRNA表达,雄激素能诱导AR　mRNA表达。小结：ER和AR蛋白及mRNA表达水平与血浆相关激素水平呈正相关。试验Ⅳ、雌、雄激素对鸡成骨细胞增殖分化、凋亡及细胞周期的影响目的：研究不同浓度雌激素和雄激素单独以及联合应用对体外培养鸡胚额骨成骨细胞增殖分化以及细胞周期和细胞凋亡的影响。方法：应用酶消化法获取15日龄鸡胚额骨成骨细胞,以雌激素和雄激素单独以及联合处理成骨细胞,MTT法比较细胞增殖率,PNPP法检测AKP变化,流式细胞仪检测细胞凋亡和细胞周期的变化。结果：雌激素和雄激素对成骨细胞增殖有一定的促进作用,雌激素(100 pg/mL)联合雄激素(100pg/mL)应用在24 h促进成骨细胞增殖作用最强,促进其分化；雌激素(200pg/mL)促进了细胞周期进程,使S期细胞显著增多；雄激素(100pg/mL)能诱导成骨细胞的凋亡；雌、雄激素联合应用使S期和G2/M期细胞增多,抑制成骨细胞凋亡。小结：雌、雄激素联合应用能够促进成骨细胞的增殖和分化,促进成骨细胞细胞周期进程,抑制细胞凋亡。试验V、雌、雄激素对鸡成骨细胞雌激素受体、雄激素受体mRNA表达目的：研究雌激素和雄激素对鸡胚额骨成骨细胞雌激素受体(ER)、雄激素受体(AR) mRNA表达水平的影响。方法：运用实时荧光定量PCR法检测鸡胚额骨成骨细胞中ER. AR mRNA的定量表达。结果：,雌激素单独应用能上调ER mRNA的表达,抑制AR mRNA的表达；雄激素单独应用对ER和AR mRNA表达无显著影响；雌、雄激素联合应用能上调ER和AR mRNA的表达。小结：雌、雄激素能够诱导相关受体的基因表达水平。更多还原

【Abstract】 Estrogen and androgen play important roles in bone metabolism. There are a lot of medullary bone formation before the sex maturation, and the medullary bone provide calcium for the calcification of the egg shell during the laying period. Estrogen and androgen are relative to the medullary bone formation, and effect osteoblasts proliferation, differentiation, cell cycle and apoptosis via estrogen receptor and androgen receptor. Considering that estrogen and androgen’s important role in medullary bone formation, there were two aims of this study, one was to find the effects of estrogen and androgen on medullary bone, and the other was to find the changes of osteoblasts function to provide theory for medullary bone formation.Experiment I Effects of Estrogen, Androgen and Both Combined on Bone Metabolism of ISA Hens before Sexual MaturityObjective: To observe the effects of estrogen, androgen and their combination on bone metabolism in the period of adolescent cage layer hens. Methods: 300 adolescent ISA hens (92 days old) were divided into 5 groups, the experiment lasted 20 days. Cortical bone histomorphometric analysis were observated. Plasma calcium (Ca), phosphorus (P), alkaline phosphatase (AKP), tartrate-resistant acid phosphatase (StrACP) were measured by automatic biochemistry analyzer. Results: Compared with control, the concentration of AKP and humerus weight increased significantly (P<0.05) in estrogen group; the concentration of AKP, humerus weight, CAR, Cr. ED and Cr. Ar increased significantly (P<0.05) in androgen group; the concentration of AKP and Ca, humerus weight, CAR, and Cr. Ar increased significantly (P<0.05) in their combined group at 10 days. The concentration of Ca and P increased (P<0.05) and the concentration of StrACP decreased (P<0.05) in estrogen group; Cr. Wi increased (P<0.05) in combined group at 15 days. Cr. ID increase and Cr. ED decrease (P<0.05) in combined group at 20 days. There was no significant difference of the control and solvent. Conclusion:It revealed that estrogen could stimulate bone formation, inhibit bone resorption and improve the bone development in hens; androgen could increase cortical bone size and mass in pre-sexual maturation hens; estrogen and androgen combined could promote bone formation, resorption, and bone turnover. ExperimentⅡEffects of Estrogen, Androgen, and Both Combined on the Medullary Bone FormationObjective: To observe the effects of estrogen, androgen, and their combination on the medullary bone formation in young birds. Methods: 300 ISA pullets and 90 ISA roosters of 92 days old were fed for one week before experiment.300 ISA pullets were averagely divided into A, B, C, D, and E groups at random, respectively. Group F was castrated ISA roosters. The experiment lasted for 20 days. In the initial and different phases of the experiment, the bone samples were collected for making bone slice. Results: The results showed that the medullary bone did not form in group A, B, or D. However, the medullary bone began to be reduced in group C at 15 days. A large amount of the medullary bone could be found in group E and F at 10 days. Compared with control A and B, the concentration of E2 increased and testosterone decreased in group C and E. (P<0.05); the concentration of E2 decreased and testosterone increased in group D (P<0.05); the concentration of E2 increased and testosterone decreased in group F (P<0.05). Conclusion:estrogen and androgen had dependent effects on medullary bone formation in pre-sexual maturation hens.ExperimentⅢEffects of the Estrogen, Androgen, and Both Combined on the Estrogen Receptor, Androgen Receptor and Their mRNA Expression in Bone Tissue in Layers.Objective: To observe the location of estrogen receptor and androgen receptor in bone tissue, and the effects of estrogen, androgen, and both combined on the estrogen receptor and androgen receptor mRNA expression. Methods: Immunohistochemistry assay was used to observe the location of estrogen receptor and androgen receptor. RT-PCR assay was used to determine the expression of estrogen receptor and androgen receptor. Results: The results of Immunohistochemistry indicated that estrogen receptor was in cell nucleus and cytoplasm of osteoblasts and osteoclasts, and increased by estrogen and both combined. The results of RT-PCR assay indicated that estrogen induce ER expression and androgen induce AR mRNA expression. Conclusion:estrogen and androgen could prompt the ER and AR expression, respectively. The ER and AR level had direct correlation with blood plasma estrogen and androgen.ExperimentⅣEffects of Estrogen, Androgen, and both Combined on Chicken Osteoblasts Proliferation, Differentiation, Cell Cycle and ApoptosisObjective: The objective of this experiment was to investigate the effects of estrogen, androgen and both combined on chicken osteoblasts proliferation, alkaline phosphatase (AKP) activity, the cell cycle, and apoptosis. Methods: The osteoblasts were treated with estrogen and androgen alone or in combination. The cell proliferation were detected with MTT method, the cell apoptosis and cell cycle with flowcytometry. Results: The results indicated that the estradiol, testosterone and both treatments could significantly enhance osteoblastic proliferation and AKP activity, accelerate the osteoblast cell cycle, and stimulated osteoblast DNA synthesis in a period of 24 h. Estrogen, used alone or with testosterone, inhibited chicken osteoblast apoptosis. However, testosterone alone induced cell apoptosis. Conclusion:Estrogen combined with testosterone promoted osteoblast proliferation and AKP activity, accelerated the osteoblast cell cycle, inhibited osteoblast apoptosis.ExperimentⅤEffects of Estrogen, Androgen, and both Combined on Chicken Osteoblast mRNA Expression of Estrogen Receptor and Androgen Receptor.Objective: The objective of this experiment was to investigate the effects of estrogen, androgen, and both combined on osteoblast mRNA expression of estrogen receptor (ER) and androgen receptor (AR). Methods: The osteoblasts were treated with estrogen and androgen alone or in combination. The mRNA expression of estrogen receptor and androgen receptor with fluorescence real-time quantitative RT-PCR. Results: Estrogen alone and combined with androgen could promote the mRNA expression of estrogen receptor; androgen and combined with estrogen could also promote the mRNA expression of androgen receptor. Conclusion:Estrogen combined with androgen could induce ER and AR mRNA expression.更多还原