臺灣博碩士論文加值系統

雌激素（estrogen）具有多種且重要的生物性效能，這些效能主要是透過與雌激素受體（estrogen receptor）的結合而產生，雌激素受體屬於細胞核受體，可以傳遞細胞外的訊息至細胞內並產生轉錄調控反應。雌激素的缺乏會造成骨質的流失，但雌激素受體在骨細胞的表現和生理調節機轉仍未完全明瞭。利用基因專一性引子以及反轉錄聚合反應，探測雌激素受體的表現，可以在人類骨癌細胞（human osteosarcoma）株MG63及HOS、人類乳癌細胞（human breast cancer cell）株MCF-7和初級培養人類類造骨細胞（human osteoblasts-like cell）得到雌激素受體α 345bp cDNA片段以及雌激素受體β 393bp cDNA片段，此外，利用雌激素受體β專一性引子進行反轉錄聚合反應時，還可在MG63、HOS、MCF-7細胞株及初級培養人類類造骨細胞發現一段220bp的cDNA片段，經由基因定序和序列比對後，證實其為一雌激素受體β變異形，缺少了雌激素受體βexon2（173bp）的序列，將此雌激素受體β變異形稱為ERβΔ2。此外，進一步利用反轉錄聚合反應探測雌激素受體α、雌激素受體β以及ERβΔ2在培養5-25天的人類骨癌細胞株MG63及HOS和初級培養人類類造骨細胞的表現。結果顯示，骨癌細胞MG63、HOS在不同天數的培養下，雌激素受體α都無表現，雌激素受體β的表現會隨著培養天數的增加而增加，在初級培養人類類造骨細胞上，所探測的3個檢體中，測出的雌激素受體α及雌激素受體β表現量有所不同，但雌激素受體β的表現明顯低於雌激素受體α，ERβΔ2在骨癌細胞HOS、MG63以及初級培養人類類造骨細胞表現量以及表現的時間不定。由於人類類造骨細胞雌激素受體β表現相對於雌激素受體α明顯偏低，而骨癌細胞中雌激素受體β表現卻偏高，因此雌激素受體β表現是否為癌化指標，值得繼續探討。

謝誌
縮寫表
中文摘要...................................................................................................1
壹、序論....................................................................................................2-8
一、骨再塑（bone remodeling）作用………………………………2
二、骨質疏鬆症[osteoporosis]………………………………………3-4
三、雌激素對骨骼的作用…………………………………………4
四、雌激素的作用機制……………………………………………5
五、雌激素受體簡介………………………………………………5-7
六、研究動機………………………………………………………8
貳、實驗材料與方法..............................................................................9-32
一、材料……………………………………………………………9-18
二、方法....................................................................................................19-32
A、細胞總量RNA之抽取…………………………………………19-20
B、反轉錄-聚合連鎖反應………………………………………20-21
C、DNA洋菜膠體電泳法…………………………………………22
D、DNA分子之萃取………………………………………………23
E、DNA接合………………………………………………………23
F、質體轉型………………………………………………………23-24
G、微量質體DNA抽取……………………………………………25
H、西方墨點法……………………………………………………25-28
I、定量反轉錄-聚合連鎖反應…………………………………28-32
參、結果..................................................................................................33-36
一、雌激素受體mRNA在乳癌細胞，骨癌細胞以及初級培養人類類造骨細胞表現之探測…………………………33
二、雌激素受體β變異型之發現及確定.........................................34
三、雌激素受體α、雌激素受體β及其變異型mRNA在初級培養人類類造骨細胞和骨癌細胞HOS、MG63培養不同天數後表現量之分析............................................................................35-36
肆、討論..................................................................................................37-39
一、雌激素受體mRNA在骨細胞的表現..........................................37
二、雌激素受體α與雌激素受體β之mRNA在初級培養人類類造骨細胞的表現……………………………37-38
三、雌激素受體變異型- ERβΔ2的發現…………………………38-39
伍、參考文獻………………………………………………………40-44
陸、附圖
附圖一.....................................................................................................45
附圖二....................................................................................................46.
附圖三..................................................................................................... 47
附圖四.....................................................................................................48
附圖五.....................................................................................................49
附圖六.....................................................................................................50
附圖七.....................................................................................................51
附圖八.....................................................................................................52
附圖九.....................................................................................................53
附圖十.....................................................................................................54
附圖十一.................................................................................................55
附圖十二.................................................................................................56
附圖十三.................................................................................................57
附圖十四.................................................................................................58
附圖十五.................................................................................................59
附圖十六.................................................................................................60
附圖十七.................................................................................................61
附圖十八.................................................................................................62
附圖十九.................................................................................................63
附圖二十................................................................................................64
附圖二十一.............................................................................................65
附圖二十二.............................................................................................66
附圖二十三.............................................................................................67
附圖二十四.............................................................................................68
附圖二十五.............................................................................................69
附圖二十六.............................................................................................70
附圖二十七.............................................................................................71
附圖二十八.............................................................................................72
附圖二十九.............................................................................................73
附圖三十.................................................................................................74

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