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研究生:張哲銘
研究生(外文):Je-Min、Chang
論文名稱:探討雄性素和雄性素接受體在小鼠骨化和造骨細胞活性所扮演的角色
論文名稱(外文):The Roles of Androgens and Androgen Receptor on Bone Formation and Osteoblast Activity
指導教授:許岩得
指導教授(外文):Yan-Der Hsuuw
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:畜產系
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:53
中文關鍵詞:雄性素接受體生長板造骨細胞
外文關鍵詞:Androgen receptorGrowth plateOsteoblast
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雄性素接受體為核膜接受體家族之一,經與雄性素結合後對骨骼生長、代謝及雄性的生殖系統發育扮演著重要調控機制。本研究目的為探討雄性素接受體(androgen receptor、AR)在小鼠骨骼發育上所扮演的角色。試驗中之小鼠飼養在獨立吹塵式無菌動物籠架,經基因型判定後,分為野生型(wild-type、WT)和雄性素接受體剔除(androgen receptor knockout、ARKO)小鼠作為主要實驗動物。在試驗一中,測量4、7、15、20、30、40和50週齡小鼠之股骨和脛骨長度,試驗結果顯示性成熟前(4或7週齡),ARKO小鼠之股骨和脛骨長度與WT小鼠並無差異;待性成熟後,顯示ARKO小鼠之股骨於20週齡後、脛骨於15週齡,其長度有逐漸比WT小鼠較長的趨勢。另一方面,以組織切片方法比較ARKO和WT小鼠生長板上軟骨細胞發育。在性成熟前WT小鼠之股骨和脛骨生長板上軟骨細胞數比ARKO小鼠多,但是性成熟後(15週齡以後),ARKO小鼠之股骨和脛骨生長板上軟骨細胞數比WT小鼠多,進一步分析在性成熟前4或7週齡股骨和脛骨的生長板軟骨和骨幹,ARKO小鼠會顯著降低股骨和脛骨生長板高度,同時初級海綿區高度和次級海綿區骨小樑數目或體積也顯著降低(P < 0.05),另一方面也顯著降低脛骨和股骨生長板高度,且降低增殖區和肥大區的軟骨細胞數(P < 0.05)。在試驗二、利用三維結構分析儀,分析7週齡WT和ARKO小鼠脛骨三維結構,其骨幹中骨量容積比、表面積比和骨小樑數目,結果顯示WT比ARKO來的高(P < 0.05),然而在骨小樑間隙,其WT比ARKO小鼠來的緻密(P < 0.05)。在試驗三,利用即時定量聚合酶連鎖反應(Real-time PCR),分析7週齡的末端股骨和近端脛骨生長板鹼性磷酸酶、Indian Hedgehog 和PTHrPR基因的表現,結果顯示Ihh、PTHrPR和ALP的mRNA表現,結果顯示ARKO小鼠比WT來的低。因此ARKO小鼠在軟骨細胞形成、分化和礦化上比WT差。在試驗四,利用小鼠頭蓋骨組織培養,評估骨的形成。我們以乳鼠頭蓋骨模式分析二氫睪固酮(dihydrotestosterone、DHT)對造骨細胞生長的影響,將4日齡ARKO和WT乳鼠的頭蓋骨分別放置在含有DHT培養液中培養7天。結果顯示,DHT的添加對WT乳鼠頭蓋骨中造骨細胞數量、增生寬度和新生骨質區域,具有刺激增生(P < 0.05),但對ARKO乳鼠的造骨細胞則無顯著影響。在試驗五,另一方面將ARKO和WT乳鼠頭蓋骨中所分離之造骨細胞分別培養在含有DHT培養液,進行造骨細胞遷移和貼附能力之分析。結果顯示,DHT對WT乳鼠頭蓋中造骨細胞的遷移和貼附能力均有顯著的增加(P < 0.05),但對ARKO乳鼠頭蓋中造骨細胞則無影響。綜合以上研究之結果,指出剔除雄性素接受體導致股骨和脛骨的生長板結構、骨幹中骨小樑數目和體積改變,雖然ARKO對發身後小鼠的股骨、脛股的長度有增長的趨勢,但此現象係因雄性素無法關閉生長板之生長導致其軟骨細胞數增加有關。由於ARKO小鼠生長板中軟骨增殖帶分化成肥大軟骨能力較差,進而造成其骨小樑數量減少;此現象與ARKO小鼠後續易罹患骨質疏鬆症有關。ARKO小鼠在軟骨細胞分化和礦化的基因表現,也發現表現較少,再者雄性素接受體的剔除亦導致骨細胞對DHT的不反應,造成骨細胞之生長、遷移、貼附能力均顯著的受到侷限。因此雄性素接受體對小鼠骨骼的生長發育與結構具有極重要之生理角色。
Androgen receptor (AR)、a member of the nuclear receptor superfamily、has been demonstrated to be important in the mediation with androgen action for the bone growth and metabolism. The aim of this study is to examine the effects of AR on the mice bone development. All study mice were maintained in a barrier facility in accord with individual ventilation cage system. All mice were determined genotypically and classified into wild type (WT) and androgen receptor knockout (ARKO) mice. The study was divided into five objectives. In objective one、we measured the length of mice femur and tibia at 4、7、15、20、30、40 and 50 weeks of age. Results show that、in comparison of femur and tibia length between ARKO and WT mice、there is no significant difference before sexual maturity (age of 4 and 7 weeks). However、the lengths of femur of ARKO after 20 weeks of age、and tibia after 15 weeks of age were trend longer than WT mice. On the other hand、we utilized the histological sections to compare the condrocytes development in growth plate between ARKO and WT. Before sexual maturity (4 and 7 weeks old)、the number of condrocytes in growth plat in femur and tibia of WT mice were increase than ARKO . However、the number of condrocytes in growth plate in femur and tibia of ARKO mice were higher than WT mice post maturity (after age 15 weeks) . Furthermore、we examined the cartilage and metaphyseal in the growth plate of femur and tibia at age of 4 and 7 week old. In the metaphysic、AR inactivation resulted in a significant reduction of growth plate height from the femur and tibia in male ARKO mice. ARKO mice showed a significant reduction in primary spongiosa height、sepongiosa trabecular bone number and secondary spongiosa trabecular bone volumes(P<0.05). In the proximal tibia and distal femur of ARKO mice、there was a significant decline in the total growth plate height、the cell number of proliferative chondrocytes per column、the cell number of hypertrophic chondrocyte per column、and the height of the resting and poliferative chondrocyte(P<0.05). In objective two、Three-dimensional computed tomography was used to exam the anatomical tibia structure of 7 weeks old WT and ARKO mice. The radiography shows that WT mice had higher percentage of bone volume、percentage of bone surface and the number of trabecular number than ARKO mice (P<0.05). However、the density of bone volume、the WT mice is more dense than ARKO mice (P<0.05). In objective three、we utilize Real-time PCR to examine the expression of Alkaline phosphatase、Indian Hedgehog and PTHrPR in growth plate in distal femur and proximal tibia of 7 weeks old both WT and ARKO mice. The results indicate that the ARKO had lower expression of Ihh、PTHrPR and mRNA in ALP than WT mice. Therefore the WT mice had better condrocytes formation、differentiation and mineralization than ARKO mice. In objective four、in order to evaluate the bone formation、the mice calvariae was used.We used the mouse calvariae organ culture model to analyze the effects of dihydrotestosterone (DHT) on the development of osteoblast.
We obtained the 4 days old ARKO and WT neonatal rat calvaria and plated separated in contain with 10nM DHT medium and maintained for 7 days.The results show that with add DHT in culture medium promote the number of osteobalst、the wildth and new bone area in WT mice (P<0.05). However、the result was no significant of ARKO mice. In objective five、in the examination the capacity of osteoblast migration and adhesion、the results indicate that the DHT increase the capacity of osteoblast migration and adhesion significantly in calvaria cell of WT mice (P< 0.05). However、no significant results in calvaria cells of ARKO mice.In conclusion、this study indicate that with knockout of androgen receptor can lead to changes in the structure of growth plate、the number of trabecular and volume of femur and tibia. Although、knockout androgen receptor may have potential effects on increase the length of femur and tibia in the post-maturity mice. However、this could due to the androgen is unable to switch the growth of growth plate which proliferate the number of condrocytes. This could be due to the low capacity of proliferation Zone differentiate to hypertrophic in growth plate consequently、the number of trabecular is decreased in ARKO mice、and it may explain the high osteoporosis in ARKO mice. Moreover、ARKO mice has less gene expression in differentiation and mineralization of condrocytes and irresponsive to DHT leading decreased capacity in the formation、migration and adhesion of calvariae cell. Therefore、the androgen receptor plays an important role in mice bone formation and morphology development.
目錄
中文摘要 I
英文摘要 III
誌VI
目錄 VII
圖表目錄 IX
壹、前言 1
貳、文獻回顧 3
ㄧ、文獻回顧 3
二、雄性素的來源 5
三、雄性素的生理功能 5
四、雄性素的代謝 6
五、雄性素接受體在生長板的表現 8
(一)生長板的軟骨細胞 8
(二)造骨細胞 8
六、骨質疏鬆症簡介 9
(一)骨骼重塑 10
(二)骨質疏鬆症的機制 12
三、材料與方法 13
一、實驗設計流程 13
(一)試驗動物 14
(二)建立頭蓋骨細胞 14
二、基因型分析 16
三、骨組織切片 19
(一)定量骨組織 19
四、三維結構分析儀 20
五、即時定量聚合酶連鎖反應分析 21
(一)步驟一:萃取檢體RNA 21
(二)步驟二:將RNA轉成cDNA 22
(三)步驟三:即時定量聚合酶連鎖反應分析 22
六、分析小鼠頭蓋骨新骨質形成 22
七、細胞行為分析 23
(一)造骨細胞細胞貼附能力分析 23
(二)造骨細胞細胞遷移能力分析 25
八、資料分析與統計 25
肆、結果與討論 26
一、探討在4週齡雄性素接受體剔除小鼠對骨幹和生長板的影響 26
二、探討在7週齡雄性素接受體剔除小鼠對骨幹和生長板的影響 29
三、探討雄性素接受體剔除小鼠對骨組織長度和生長板細胞數的影響 32
四、探討雄性素接受體剔除小鼠在骨組織三維結構中骨小樑變化 34
五、探討雄性素接受體剔除小鼠在骨組織基因的表現 37
六、探討雄性素接受體剔除小鼠頭蓋骨對新骨質形成的影響 39
七、探討雄性素接受體剔除的頭蓋骨細胞行為之影響 41
(一)探討雄性素接受體剔除的頭蓋骨細胞對貼附能力之影響 41
(二)探討雄性素接受體的剔除頭蓋骨細胞對遷移能力之影響 41
伍、結論 45
六、參考文獻 46
作者簡介 50
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