臺灣博碩士論文加值系統

牙周疾病所造成的骨缺損，隨著嚴重程度的增加，會造成牙齒穩定度改變而動搖，所以當今口腔醫學界努力研究的目標，就是希望補救此種骨缺損並恢復正常的生理結構及功能。
理想中好的骨移植材料，是能夠誘導骨質與牙骨質的生長，並可以促進牙周組織有新的附連產生（new attachment），另外此種骨移植材料最好是容易取得、容易操作、不昂貴且有很好的生物相容性，以及沒有潛在性疾病傳染的危險性存在。
從原始間質細胞分化而來的造骨細胞，其細胞的分化，會參與骨質的分泌和鈣化，所以對於新骨的形成扮演著重要的角色。
微脂粒為人工合成具有雙層磷酸脂分子，可以形成鈣化的核心，並促進類骨質細胞生長的作用。目前微脂粒已被廣泛應用於藥物傳遞的系統，並作為生物包膜模型的研究。先前我們的研究已經證實酸性負電荷的微脂粒含磷酸脂絲氨的濃度越高（100μmol/L），對於造骨細胞礦物質化的促進有正向的作用。
在牙周疾病的治療中，經常會使用的抗生素即是四環黴素，目前我們知道四環黴素可以用作牙周保守性處理時，局部沖洗牙齦下囊袋的藥物；牙周手術治療時牙根面處理的藥劑；也可以抑制宿主產生膠原蛋白酶來破壞牙周組織。
實驗中造骨母細胞取自Sprague-Dawley品係大白鼠21天大的初代培養胚胎顱骨細胞，在35mm培養皿中加入由同一成分的負電荷酸性微脂粒（egg phosphatidylcholine, cholesterol, bovine brain phosphatidylserine）包埋不同濃度的四環黴素來作為比較，探討不同濃度的四環黴素被微脂粒包埋所產生的作用為何。實驗所用的比較方法分成增生組【計算細胞數目：分別取24hr、48hr、72hr、96hr的結果作比較；測定鹼性磷酸酶:分別取8天、12天、16天、20天的結果作比較】及鈣化組【以von Kossa’s stain染鈣化組織：分別取12天、16天、20天、24天的鈣化點數作為比較】。
實驗的結果顯示，含有負電荷的酸性微脂粒所包埋不同濃度的四環黴素，對於造骨細胞的細胞增生情形沒有顯著的影響;而在各組鈣化點數的比較，負電荷的酸性微脂粒及其所包埋不同濃度的四環黴素，與控制組相較，則有顯著性的增加，並以ANOVA統計分析所產生的結果，此顯著性的增加具有統計學上的意義（p<0.0001）。目前已經知道負電荷的酸性微脂粒可以增加造骨細胞的骨質鈣化，而加上四環黴素包入微脂粒中，更有加成的效果，可以促進鈣化組織的形成，進而加速類骨質組織的產生。
含有磷酸脂絲氨的微脂粒，所包埋不同濃度的四環黴素中（0, 0.065, 0.13, 0.26μg/ml），當四環黴素濃度愈高，所產生的鈣化點數目越多，統計結果是具有統計學上的意義(p<0.0001)。

The major aim of therapies for bony defects is promoting new bone formation. Osteoblasts, differentiated from primitive mesenchymal cells, play an important role in bone formation, including cell proliferation, synthesis, bone matrix secretion and calcification.
Previous studies indicated that mineralization in bone is associated with lipids, particularly phospholipids which enriched in cell-dervied matrix vesicles. They serve as nuclei for new crystals formation. Liposomes are artificial microscopic vesicles composed of bimolecular phospholipid layers. They are useful membrane models for the study of biological mineralization.
Our previous investigation had demonstrated that liposomes composed of bovine brain phosphatidylserine(BBPS) could promote calcification in rat osteoblast-enriched cultures. On the other hand, tetracycline(Tc) is a useful therapeutic agent in periodontal treatment, which may have influence on the process of new bone formation.
In the present study, we would like to determine whether tetracycline embedded in BBPS liposomes have strong affinity for calcium and apatite mineral deposition.
The purpose of the experiment was to observe the proliferation of osteoblast-like cells by measuring cell numbers, alkaline phosphatase(ALP) activity, and calcified particles stained by von Kossa’s method. The dose dependency and time effect of liposomes on cell number; ALP activity and calcified particles were analyzed by two-way ANOVA and one-way ANOVA. Individual comparisons between pairs were examined by two-sample t-test.
Effects on cell proliferation in embedded and nonembeded groups were not significantly different. The elevation of ALP activity in the cells was dramatic from day 12 to day 16 in test group, and this change with time was statistically significant (p<0.0001). Tetracycline embedded liposomes significantly reduced ALP activity (p<0.0001), and there was a dose dependent effect of tetracycline. The number of calcified particles in tetracycline embedded group was significantly greater than nonembedded group (p<0.0001). Dose dependent effect of tetracycline was noted.
These findings suggest that tetracycline embedded liposomes can function like matrix vesicles to induce the formation and calcification of bone-like tissue. In addition, tetracyclines embedded liposomes do not reduce the expression of osteoblast-like cells or the cell growth in primary rat osteoblast-enriched cultures.

第一章、 摘要………………………………………… 1
第二章、 英文摘要…………………………………… 4
第三章、 前言………………………………………… 7
第一節、 研究背景及動機…………………………………7
第二節、 研究目的………………………………………10
第四章、 文獻回顧…………………………………… 12
第一節、 基質囊泡與骨質鈣化……………………………12
第二節、 微脂粒的基本結構與組織………………………15
第三節、 微脂粒與鈣化機制的研究…………………………17
第四節、 微脂粒的應用與微脂粒作為藥物傳遞工具…………19
第五節、 四環黴素的探討…………………………………20
第六節、 造骨細胞培養方法的研究…………………………24
第五章、 材料與方法………………………………… 26
第一節、 培養液的製備…………………………………26
第二節、 取骨片的方法…………………………………26
第三節、 細胞培養的方法………………………………27
第四節、 微脂粒的製備方法與程序…………………………29
第五節、 實驗分組………………………………………31
第六節、 細胞數目的計算方法……………………………31
第七節、 鹼性磷酸酶的活性測定方法………………………32
第八節、 鈣化染色的方法…………………………………35
第九節、 資料處理與分析…………………………………37
第六章、 結果………………………………………… 38
第一節、 造骨細胞在顯微鏡下的生長形態觀察………………38
第二節、 計算造骨細胞的增生數目…………………………39
第三節、 造骨細胞鹼性磷酸酶活性的測定結果………………45
第四節、 計算造骨細胞的鈣化點數目………………………51
第七章、 討論………………………………………… 57
第八章、 結論………………………………………… 62
第九章、 參考文獻…………………………………… 64
第十章、 附表………………………………………… 69
第十一章、 附圖……………………………………… 109

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