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研究生:賴冠達
研究生(外文):Lai, Kungda
論文名稱:利用摘除卵巢小鼠為動物模式評估克弗爾延緩骨質流失之功效
論文名稱(外文):The Functional Assessment of Kefir on thePostponement of Bone Loss in Ovariectomized Mice
指導教授:陳小玲陳小玲引用關係
指導教授(外文):Chen, Hsiaoling
口試委員:黃尉東謝長奇
口試委員(外文):Huang, WeitungHsieh, Changchi
口試日期:2011-07-28
學位類別:碩士
校院名稱:大葉大學
系所名稱:分子生物科技學系碩士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:90
中文關鍵詞:摘除卵巢小鼠克弗爾Micro-CT掃描式電子顯微鏡
外文關鍵詞:ovariectomized micekefircomputerized tomographyscanning electron microscopy
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停經後婦女較易產生骨質疏鬆,主因為卵巢失去功效無法分泌雌激素與異黃酮,導致無法促進成骨細胞生長以及抑制破骨細胞過度活躍之能力因而造成骨質疏鬆。前人研究酪蛋白磷酸肽具有與鈣結合之能力,且具有易吸收與 延緩鈣質與骨質流失之功能。本試驗即是以去卵巢小鼠作為模擬女性停經後之動物模式,評估發酵乳製品-克弗爾延緩骨質流失之功效。本試驗共分兩階段:(1) 克弗爾在摘除卵巢老齡鼠之骨質流失延緩功效評估。本試驗以八周齡之B6小鼠進行摘除卵巢,做為模擬女性停經後因激素缺乏導致骨質疏鬆症之動物模式,而後分五個處理組,分別為切除卵巢處理組 (Water/OVX)、假手術處理組 (Water/Sham)、碳酸鈣補充組 (Ca/OVX)、克弗爾補充組 (Kefir/OVX) 及克弗爾加上碳酸鈣補充組 (Kefir+Ca/OVX) 。各組餵養至16月齡後犧牲,並以Micro-CT分析膝蓋端生長板骨小樑。結果顯示,於膝蓋端生長板骨小樑之3D立體結構、骨佔組織比例 (BV/TV) 、骨小樑骨礦物密度 (BMD) 、骨小樑厚薄度 (Tb.Th) 、骨小樑數量 (Tb.N) 、骨小樑分離度 (Tb.Sp)及至密骨骨礦物密度 (BMD) 皆未有明顯差異。推論造成此情形的原因可能是鼠齡過老,即使餵有營養補充功效之補充劑亦無法延緩骨質流失 。(2) 克弗爾在摘除卵巢小鼠模擬女性中年更年期的骨質流失延緩功效評估。本試驗以達到性成熟之4個月齡小鼠進行摘除卵巢,做為模擬女性停經後因激素缺乏導致骨質疏鬆症之產生,同樣分為五種處理組餵養2個月後犧牲,並以Micro-CT來分析膝蓋端生長板骨小樑得知,膝蓋端生長板骨小樑之3D立體結構組間即有顯著性差異,骨佔組織比例 (BV/TV) Kefir/OVX組比Water/OVX組上升75%、骨小樑骨礦物密度 (BMD) Kefir/OVX組比Water/OVX組上升27%、 (Tb.Th) Kefir/OVX組比Water/OVX組上升24%、 (Tb.N) Kefir/OVX組比Water/OVX組上升55%而 (Tb.Sp) Water/OVX組比Kefir/OVX組上升18%,統計後結果顯示皆以Kefir/OVX處理組為五種處理組中骨小樑維持量最高者。此試驗結果證實克弗爾具有延緩骨質流失之效果。
Postmenopausal women would be more prone to osteoporosis, because the ovary was atrophied and unable secret enough estrogen and isoflavones, which can promote the growth of osteoblasts and inhibit the activity of osteoclast. In previous studies, casein phosphopeptides (CPPs) were found that exhibit the ability to bind calcium and to enhance calcium absorption into the body. CPPs may be the functional materials to postpone the bone loss. Kefir is a kind of fermented milk with various bioactivities reported in many previous research papers. However, there is few osteoporosis-related papers published. Therefore, this study was conducted to verify the bioactivity of osteoporosis prevention in kefir. Experiment were divided into two part (1) the assessment of kefir on the postponement bone loss in the old aged ovariectomied mice. Eight-week-old B6 ovariectomized mice was used to be the animal model to simulate menopause women. There were five treatment, including ovariectomized groups (Water / OVX), sham groups (Water / Sham), calcium and carbonate supplement groups (Ca / OVX), kefir supplement groups (Kefir / OVX) and kefir combined with calcium carbonate supplement groups (Kefir + Ca / OVX). Mice were sacrificed after 16 months treatment. Micro-CT was used to analyze trabecular bone in the knee growth plates. Result showed that three-dimensional structure, proportion of bone volume/tissue volume (BV / TV), trabecular bone mineral density (BMD), trabecular thickness degree (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb. Sp) and cortical bone density (BMD),did not had significant difference between various treatments. (2) the assessment of kefir on the postponement bone loss in the ovariectomied mice. Four-month old sexual matured and ovariectomied mice were used. There were five treatment as described above. Mice were sacrificed after 2 months treatment. The results of Micro-CT showed that the Kefir / OVX group was significantly different compared with the other group in three-dimensional structure of trabecular bone in knee growth plate. In the proportion of bone tissue and bone volume/tissue volume (BV / TV), Kefir / OVX was about 75% higher than Water / OVX ,group. In trabecular bone mineral density (BMD), Kefir / OVX group was 27% higher than Water / OVX group. In trabecular bone thickness (Tb.Th) and trabecular bone number (Tb.N), Kefir / OVX group was 24% and 55% higher than Water / OVX group. In trabecular bone separation (Tb.Sp), Water / OVX group was 75% higher than Kefir / OVX group. In addition, the result of SEM showed that the Kefir / OVX group was higher then the other groups. Kefir / OVX group has the highest amount of trabecular bone in five groups. According to the results described above, kefir exhibited the ability of postponement bone loss.
目錄

封面內頁
簽名頁
中文摘要....................................................................................... iii
英文摘要....................................................................................... v
誌謝............................................................................................... vii
目錄............................................................................................... viii
圖目錄........................................................................................... ix
表目錄........................................................................................... xviii

1. 前言............................................................................................ 1
2. 文獻回顧.................................................................................... 2
2.1 骨質疏鬆症介................................................................ 2
2.2 骨骼的組成結構、生合成以及代謝…........................ 5
2.2.1 破骨細(Osteoclast)................................................. 6
2.2.3 骨細胞 (Osteocytes)................................................. 9
2.2.4 骨吸收(Bone resorption)...................................... 10
2.2.5 骨生成 (Bone remodeling)....................................... 10
2.3 動物模式.......................................................................... 12
2.4 分析儀器-掃描式電子顯微鏡 (Scanning Electric Tomography:SEM)..................................................................
13
2.4.1 分析儀器-電腦斷層掃描…................................... 13
3. 材料與方法….............................................................................. 16
3.1 實驗架構規畫.................................................................. 16
3.2材料方法 (一)…............................................................... 16
3.2.1 (一) 克弗爾在摘除卵巢老齡鼠的骨質流失延緩功效評估.................................................................................
16
3.2.1.1 小鼠來源..........................................................
16
3.2.1.2 動物試驗分組.................................................. 17
3.2.1.2.1偽手術組.................................................. 17
3.2.1.2.2切除卵巢組............................................... 18
3.2.1.2.3營養補充組.............................................. 18
3.2.1.2.4克弗爾來源及劑量.................................. 18
3.2.1.3鼠骨試片樣本處理及分析…...…...…….......... 19
3.2.1.3.1 次氯酸鈉處理......................................... 19
3.2.1.3.2 脫水處理................................................. 20
3.2.1.4 儀器分析.......................................................... 20
3.3.1.4.1 高解析度微型 X光電腦斷層掃瞄儀 (Micro-CT)…..........................................................
20
3.2.1.4.2 掃描式電子顯微鏡(SEM)….................. 21
3.2.2 (二) 克弗爾在摘除卵巢小鼠相對女性中年更年期
的骨質流失延緩功效評估.................................................
22
3.2.2.1 小鼠來源.......................................................... 22
3.2.2.2 動物試驗分組.................................................. 23
3.2.2.3 偽手術組.......................................................... 23
3.2.2.4 切除卵巢組...................................................... 23
3.2.2.5 營養補充組...................................................... 24
3.2.3 克弗爾來源及劑量…............................................... 24
3.2.4 分析方法................................................................... 25
3.3 統計分析.......................................................................... 25
4. 結果….......................................................................................... 26
4.1 克弗爾在摘除卵巢老齡鼠的骨質流失延緩功效評估.. 26
4.1.1 小鼠體重變化........................................................... 26
4.1.2 股骨外觀圖............................................................... 26
4.1.3 以Micro-CT分析膝蓋端生長板骨小樑的3D立體結構.....................................................................................
26
4.1.4 以Micro-CT分析膝蓋端生長板骨小樑與組織所佔之比例.............................................................................
27
4.1.5 以Micro-CT分析膝蓋端生長板骨小樑厚薄度..... 27
4.1.6 以Micro-CT分析膝蓋端生長板骨小樑數量......... 27
4.1.7 以Micro-CT分析膝蓋端生長板骨小樑分散程度. 28
4.1.8 以Micro-CT分析膝蓋緻密骨骨礦物密度............. 28
4.2 克弗爾在摘除卵巢小鼠相對女性中年更年期的骨質流失延緩功效評估….............................................................
29
4.2.1 小鼠體重變化…....................................................... 29
4.2.2 股骨外觀圖…........................................................... 29
4.2.3 以Micro-CT分析膝蓋端生長板骨小樑的3D立體結構….................................................................................
30
4.2.4 以Micro-CT分析膝蓋端生長板骨小樑與組織所佔之比例.............................................................................
30
4.2.5 以Micro-CT分析膝蓋端生長板骨小樑骨礦物密度….....................................................................................
30
4.2.6 以Micro-CT分析膝蓋端生長板骨小樑厚薄度..... 31
4.2.7 以Micro-CT分析膝蓋端生長板骨小樑數量......... 31
4.2.8 以Micro-CT分析膝蓋端生長板骨小樑分散程度. 32
4.2.9 以Micro-CT分析膝蓋緻密骨骨礦物密度............. 32
4.2.10 以掃描式電子顯微鏡觀察膝蓋端生長板骨小樑的外觀切片….....................................................................
33
4.2.11 以Micro-CT分析膝蓋端生長板骨小樑的3D縱切立體結構….....................................................................
33
4.2.12 五種處理組小鼠臟器外觀觀察…...................... 33
5. 討論….......................................................................................... 35
5.1 克弗爾在摘除卵巢老齡鼠的骨質流失延緩功效評估... 35
5.2 克弗爾在摘除卵巢小鼠相對女性中年更年期的骨質流失延緩功效評估….............................................................
36
6. 結論.............................................................................................. 39
參考文獻........................................................................................... 70

圖目錄

圖1 骨骼微結構…....................................................................... 41
圖2 成破骨細胞生長示意圖…................................................... 42
圖3 五種處理組之試驗動物處理時程…................………....... 43
圖4 五種處理組小鼠經16個月餵食後之 Micro-CT 掃描影像圖…...................................................................................
44
圖5 以Micro-CT分析軟體CTan分析膝蓋端生長板骨小樑之圈選範圍…...................……………………………………
45
圖6 五種處理組之試驗動物處理時程…..........………………. 46
圖7 五種處理組小鼠餵養16個月後之體重變化…................. 47
圖8 五種處理組小鼠餵養16個月後之股骨頭外觀圖…............................................................................….......
48
圖9 五種處理組小鼠餵養16個月後之膝蓋端生長板骨小樑Micro-CT合成圖…..............................................................
49
圖10 五種處理組小鼠餵養16個月後利用Micro-CT分析膝蓋端骨小樑與組織所佔之比例比較…...................................
50
圖11 五種處理組小鼠餵養16個月後利用Micro-CT分析膝蓋端生長板骨小樑之厚薄度比較….......................................
51
圖12 五種處理組小鼠餵養16個月後利用Micro-CT分析膝蓋端生長板之骨小樑數量比較…...........................................
52
圖13 五種處理組小鼠餵養16個月後利用Micro-CT分析膝蓋端生長板之骨小樑分離度比較….......................................
53
圖14 五種處理組小鼠餵養16個月後利用Micro-CT分析膝蓋緻密骨之骨礦物密度比較…...............................................
54
圖15 小鼠與人類年齡相對圖....................................................... 55
圖16 五種處理組小鼠餵養2個月後之體重變化…................... 56
圖17 五種處理組小鼠餵養2個月後之股骨頭外觀圖................ 57
圖18 五種處理組小鼠餵養2個月後之膝蓋端生長板骨小樑合圖….......................................................................................
58
圖19 五種處理組小鼠餵養2個月後之膝蓋端生長板骨小樑側面合成圖…...........................................................................
59
圖20 五種處理組小鼠餵養2個月後利用Micro-CT分析膝蓋端生長板之骨小樑與組織所佔之比例比較…...................
60
圖21 五種處理組小鼠餵養2個月後利用Micro-CT分析膝蓋端生長骨小樑之骨礦物密度比較…...................……........
61
圖22 五種處理組小鼠餵養2個月後利用Micro-CT分析膝蓋端生長板骨小樑之厚薄度比較….......................................
62
圖23 五種處理組小鼠餵養2個月後利用Micro-CT分析膝蓋端生長板骨小樑之數量比較…...........................................
63
圖24 五種處理組小鼠餵養2個月後利用Micro-CT分析膝蓋端生長板之骨小樑分離度比較….......................................
64
圖25 五種處理組小鼠餵養2個月後利用micro-CT分析膝蓋緻密骨骨礦物之密度比較….............……….……….........
65
圖26 五種處理組小鼠餵養2個月後利用掃描式電子顯微鏡分析膝蓋端生長板之骨小樑比較...........................................
66
圖27 五種處理組小鼠餵養2個月後之膝蓋端生長板骨小樑縱切面合成圖…..….............................................................…
67
圖28 五種處理組小鼠餵養2個月後臟器外觀圖…................... 68
表目錄

表1 世界衛生組織 (WHO) 骨質疏鬆症分級方式….............. 69

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