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研究生:黃薇蓁
研究生(外文):Huang Wei-Chen
論文名稱:不同微脂粒成份對於造骨母細胞礦物質化的影響
論文名稱(外文):Different compositions of liposomes on mineralization in rat osteoblast-enriched cultures
指導教授:黃景勝
指導教授(外文):Huang Jiing-Sheng
學位類別:碩士
校院名稱:高雄醫學大學
系所名稱:牙醫學研究所
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2000
畢業學年度:88
語文別:中文
論文頁數:90
中文關鍵詞:微脂粒基質囊泡礦物質化氫氧磷灰石
外文關鍵詞:liposomematrix vesiclemineralizationhydroxyapatite
相關次數:
  • 被引用被引用:2
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  • 下載下載:51
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摘 要
牙周病造成牙齦發炎,附著結締組織喪失,最嚴重的即是齒槽骨的缺損(alveolar bone defects)而牙齒動搖。因此期望能夠有理想的骨組織修復材料,恢復支持性骨組織並能夠改善牙周疾病為主要積極研究目的。
現今對於牙周病的治療已有相當多的研究,然而尚未有完全理想的材料;理想的骨組織修復材料必須具有生物相容性,易取得、有經濟性、長效性,且能刺激並促進新生骨組織的形成,進而達到骨質修復的能力。
微脂粒已被廣泛應用於藥物傳遞系統,及作為生物胞膜模型之研究;並且其組成與細胞膜及脊椎動物硬組織中,引起早期鈣化沉積的基質泡類似,可以作為模擬基質泡礦物質化沉積機制的研究;微脂粒也已經被證實具有促進類骨質細胞生長之作用。
微脂粒為人工合成,具有雙層磷酸脂分子,可包覆水溶液而隔絕於外界,並且可以形成鈣化之核心。其鈣化反應開始於微脂粒內成份與膜外無機磷酸鹽類作用,產生內部自體沉澱,進而引起膜外大量沉澱並且形成鈣化組織。
微脂粒具有不同成份及構造,其組成及表面電荷將會使微脂粒有不同之特性。微脂粒分為中性微脂粒及酸性微脂粒;酸性微脂粒被認為在引發和調節基質囊泡之生物礦物質化中具有重要的角色;且酸性微脂粒中又可以分成正電荷及負電荷;本實驗研究目的即是探討酸性負電荷微脂粒與中性微脂粒對於造骨母細胞礦物質化的影響。
實驗中造骨母細胞取自Sprague-Dawley品系大白鼠21天大的初代培養胚胎顱骨細胞,在35mm培養皿中加入不同成份之微脂粒,如:中性微脂粒(egg phosphatidylcholine, cholesterol)及負電荷酸性微脂粒(egg phosphatidylcholine, cholesterol, bovine brain
phosphatidylserine)作為比較,以探討不同成份微脂粒的作用;然而不同成份之微脂粒作用差異在於是否含有負電荷的酸性磷酸脂絲氨微脂粒(phosphatidylserine)。實驗將不同成份之微脂粒各分成100µmole/L及20µmole/L的不同濃度及不含任何一種微脂粒的控制組作為比較;實驗所用之比較方法分成增生組【計算細胞數目與測定鹼性磷酸酉每活性;分別取8天、12天、16天、20天之結果作比較】及鈣化組【以von Kossa''s stain染鈣化組織;分別取12天、16天、20天、24天之鈣化點數目結果作為比較】。
實驗結果顯示兩種不同成份的微脂粒,對於造骨母細胞的細胞數目增生性沒有顯著影響;至於各組鈣化點數目之比較;含有負電荷微脂粒(egg phosphatidylcholine, cholesterol, bovine brain phosphatidylserine )則有顯著性的增加,且分別以ANOVA 統計方法分析結果,則具統計學上的顯著意義(p<0.0001)。由統計結果中可見含有負電荷酸性磷酸脂絲氨微脂粒可以增加造骨母細胞的骨質鈣化,形成鈣化組織,進而加速類骨質組織的生成。
含有磷酸脂絲氨的微脂粒中,不同濃度100µmole/L及20µmole/L的不同濃度作為比較,濃度越高(100µmole/L)的微脂粒所形成之鈣化點數目越多,以two-sample t-test作統計分析,統計結果則具有統計學上的顯著意義(p<0.0001)。
Abstract
Destruction of periodontal tissues is characterized by recurrent symptoms such as gingival inflammation, followed by loss of connective tissue and alveolar bone. Many new techniques and materials have been developed in the past years, but there is still no perfect procedure for cementum, alveolar bone and gingival attachment regeneration.
Liposomes have been used as the biomembrane model for many years in the research of drug delivery. It has been proven that liposomes have the promotional ability of osteoblast-like cells. As an artificial membranous lipid vesicle, liposomes can serve as the model structure for
biological calcification, but there is no definite conclusion that liposomes can function like matrix vesicles to induce bone-like tissue formation and calcification. However, liposomes containing phosphatidylserine(PS)-giving negatively charged liposomes-can be incorporated into the nucleus of osteoblast-like cells, leading to nucleoplasm crystallization and eventual calcification of the entire cell.
The experiment included three groups: a control group, a group consisting egg phosphatidylcholine(EPC) and cholesterol (Chol) at different concentrations, and a group containing bovine brain phosphatidylserine (BBPS) with EPC and Chol at
different concentrations. Two types of liposomes, each at different concentrations, were added to the 21st-day Sprague-Dawley fetal rat calvarial cell cultures.
The purpose of the experiment was to observe the proliferation of osteoblast-like cells by measuring cell numbers and alkaline phosphatase (ALP) activity, and calcified particles stained by von Kossa’s method. The data was analyzed by one-way and two-way ANOVA. The control group and two experimental liposomes showed a significant increase in osteoblastic cell numbers between Day 4-20 and ALP activity between Day 12-16. There was, however, no significant difference in the rate of growth.
Calcified particles from Day 12-24 detected by von Kossa’s stain were larger and more abundant in the group containing BBPS, EPC, and Chol than the group consisting only EPC and Chol, or the control group (p<0.0001). The responses were dependent on the concentration of liposomes. These findings suggest that liposomes can function like matrix vesicles to induce bone-like tissue formation and calcification. However, liposomes will not reduce the expression of osteoblast-like cells or the cell growth in primary rat osteoblast-enriched cultures.
目 錄
第一章、 摘要 ------------------------------------------- 1
第二章、 英文摘要 --------------------------------------- 3
第三章、 緒論
第一節、研究背景及動機 ------------------------------- 5
第二節、研究目的 ------------------------------------- 6
第四章、 文獻回顧
第一節、微脂粒的應用與微脂粒作為藥物傳遞工具 --------- 7
第二節、微脂粒的基本結構與組織 -- -------------------- 8
第三節、微脂粒與鈣化機制之研究 ---------------------- 10
第四節、造骨母細胞培養之研究 ------------------------ 12
第五章、 材料與方法
第一節、微脂粒之製備方法與程序 ------------------------- 14
第二節、培養液之製備方法 ---------------------------- 15
第三節、取骨片之方法 -------------------------------- 15
第四節、細胞培養之方法 ------------------------------ 16
第五節、實驗分組 ------------------------------------ 17
第六節、細胞數目之計算 ------------------------------ 18
第七節、鹼性磷酸酉每之活性測定方法 ------------------ 18
第八節、鈣化染色之方法 ------------------------------ 20
第六章、 結果
第一節、顯微鏡下之觀察 ------------------------------ 22
第二節、細胞數目之計算結果 -------------------------- 22
第三節、鹼性磷酸酉每之活性測定結果 ------------------ 25
第四節、鈣化點數目之計算結果 ------------------------ 29
第七章、 討論 ------------------------------------------ 33
第八章、 結論 ------------------------------------------ 38
第九章、 參考文獻 -------------------------------------- 40
第十章、 附表 ------------------------------------------ 46
第十一章、 附圖 ---------------------------------------- 73
參 考 文 獻
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