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研究生:陳成欽
研究生(外文):Chen Chern-Chin
論文名稱:人類乳牙牙髓造纖維細胞的細胞培養及與牙髓組織鈣化有關的酵素和蛋白質的研究
論文名稱(外文):The Cell Culture of Human Deciduous Pulpal Fibroblast and the Study of Enzymes and Proteins Related to Pulp Calcification
指導教授:黃純德黃純德引用關係
指導教授(外文):Shunte Huang
學位類別:博士
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:2001
畢業學年度:89
語文別:中文
論文頁數:137
中文關鍵詞:乳牙牙髓造纖維細胞鹼性磷酸脢琥珀去氫脢細胞毒性牙科材料纖維黏連蛋白第一類膠原蛋白第三類膠原蛋白
外文關鍵詞:Deciduous pupal fibroblastalkaline phosphatasesuccinate dehydrogenasecytotoxicitydental materialsfibronectincollagen type Icollagen type III.
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乳牙的牙根具有被吸收脫落以利繼生恆牙萌出的自然生理現象, 因此其牙髓組織的生理與病理反應是否與恆牙相同? 其牙髓組織的活性及修復能力是否也跟恆牙一樣呢? 且在臨床上乳牙齲齒填補治療後常較恆牙容易引起牙髓病變, 因此乳牙與恆牙牙髓細胞與鈣化有關的酵素對填補材料的反應及牙髓細胞與鈣化有關的蛋白質含量是否相同? 所以本實驗的目的即在探討研究乳牙牙髓細胞與恆牙牙髓細胞在與鈣化有關的的酵素與蛋白質差異. 本實驗首先培養人類乳牙的牙髓造纖維細胞, 建立本實驗室的細胞株; 然後將保存在液態氮內第五至八代間之人類乳牙牙髓造纖維細胞解凍後, 以基本培養液, 培養在37℃, 5% CO2, 95% 空氣的培養箱中, 可以穩定的成長, 在7天的觀察期間內, 其生長曲線成線性關係; 並以 Pearson bivariate correlation法分析,測量乳牙牙髓造纖維細胞之琥珀去氫脢含量與細胞數目的相關性,在統計上有顯著差異 (p<0.01 level, 2-tailed), 其相關係數 (correlation coefficients) 為0.972. 所以乳牙牙髓造纖維細胞琥珀去氫脢的MTT assay 可以取代單純的計算細胞數目.
接著再測試各種牙科材料對乳牙牙髓造纖維細胞之琥珀去氫脢及鹼性磷酸脢的影響. 測試琥珀去氫脢可以瞭解材料對牙髓細胞的毒性, 測試鹼性磷酸脢可以瞭解材料對牙髓內似造牙本質細胞形成鈣化組織的能力, 經ANOVA 及 Duncan's test 分析, 其琥珀去氫脢MTT assay的結果顯示, 乳牙牙髓造纖維細胞在接觸覆髓材料時琥珀去氫脢活性的大小依次為IRM = Dycal < TimeLine < Medium; 在接觸暫時填補材料時大小依次為 IRM = ZP < PC = Medium; 在接觸填補材料時大小依次為 IRM < FujiIILC <FujiII = Z100 = Dyract < Medium. 鹼性磷酸脢ALP assay的結果顯示, 乳牙牙髓造纖維細胞在接觸覆髓材料時鹼性磷酸脢活性的大小依次為 IRM = Dycal < Medium < TimeLine; 在接觸暫時填補材料時大小依次為IRM < ZP = PC = Medium; 在接觸填補材料時大小依次為 IRM < Z100 = Medium < FujiIILC = Dyract = FujiII; 所以由此部份的結果顯示, 各種不同牙科材料對乳牙牙髓造纖維細胞的琥珀去氫脢與鹼性磷酸脢的影響並不一致, 所以如果我們要瞭解某個牙科材料對乳牙牙髓造纖維細胞真正的的毒性,我們需要利用多種不同的測試方法, 來判斷材料對乳牙牙髓造纖維細胞各種不同酵素的影響.
另外比較乳牙與恆牙牙髓造纖維細胞的琥珀去氫脢, 在接觸各種測試的牙科材料時的影響, 結果顯示材料對乳牙及恆牙均有毒性反應, 但是乳牙與恆牙間沒有顯著的差異, 而各種測試的牙科材料對乳牙與恆牙牙髓造纖維細胞鹼性磷酸脢的活性的影響則有顯著的差異, 且乳牙牙髓造纖維細胞的鹼性磷酸脢活性較恆牙牙髓造纖維細胞低, 所以在材料的毒性測驗上, 乳牙應該與恆牙分別做測試, 才可以顯現乳牙牙髓細胞的特性, 而鹼性磷酸脢是與鈣化有關的酵素, 所以乳牙牙髓組織內似造牙本質細胞在修復性牙本質的再生能力上會比恆牙來的低, 此項發現與乳牙齲齒填補治療後較容易引起牙髓病變的臨床現象一致.
最後再利用antibody-conjugated secondary antibody assay, 以恆牙牙周造纖維細胞(G)及 3T3細胞(T)當成對照組, 來比較乳牙(D)及恆牙(P)的牙髓造纖維細胞的纖維黏連蛋白,膠原蛋白I, III等與鈣化有關的蛋白質含量差異. 結果顯示各種細胞的纖維黏連蛋白 OD值大小依次為: T < D = P = G; 各種細胞膠原蛋白 I OD值大小依次為: T < D = P < G; 各種細胞膠原蛋白 III OD值大小依次為: T < P < D < G, 所以乳牙其牙髓造纖維細胞的纖維黏連蛋白與膠原蛋白I 和恆牙牙髓造纖維細胞相同, 但是乳牙牙髓造纖維細胞的膠原蛋白 III 比恆牙牙髓造纖維細胞多, 根據其他學者的研究發現, 膠原蛋白 III 的存在會使組織無法正常鈣化, 所以乳牙牙髓組織內類造牙本質細胞在生理狀態下的再鈣化,或病理狀態下修復性牙本質的再生可能會較恆牙差.
The first purpose of the study was to establish the cell line of human deciduous pulpal fibroblasts (HDPF). Then with the serially dilute method, the relationship of cell numbers of HDPF and optical density (OD) of succinic dehydrogenase (SDH) activity was analyzed by MTT assay and statistics assay by Pearson bivaritae correlation showed that the correlation was significant at the 0.01 level (2-tailed), and the correlation coefficient was 0.972. It meant that MTT assay for SDH activity could be accepted as substitute for HDPF cell number counting.
The second purpose of the study was to evaluate the responses of SDH and alkaline phosphatase (ALP) activities of HDPF to dental materials. Tested materials included Dycal (Dentsply), TimeLine (Dentsply), Z100 (3M), Dyract (Dentsply), FujiII(GC), FujiIILC(GC), Super cement (SHOFU), Hy-bond (SHOFU). IRM (Dentsply) and culture medium (MD) alone were used as positive and negative controls, respectively. HDPF (10000 cells/well) was incubated with 100ml of extract and 20 % FBS in a 96-well plate for 24 hours in a 37℃, 5% CO2 incubator. Six wells per material were prepared. Optical density of SDH and ALP of HDPF were measured by a spectrophotometer. The means were analyzed by ANOVA and then Duncan Test. The capping materials ranking of OD of SDH was IRM = Dycal < TimeLine < Medium. Its ranking of OD of ALP was IRM =Dycal < Medium < Time Line. The temporary filling materials ranking of OD of SDH was IRM = ZP < PC = Medium. Its ranking of OD of ALP was IRM < ZP < PC = Medium. The filling materials ranking of OD of SDH was IRM < FujiIILC < FujiII = Z100 = Dyract < Medium. Its ranking of OD of ALP was IRM < Z100 < Medium < FujiIILC = Dyract = FujiII. It was suggested that, in addition to vitality test, such as SDH activity, crucial function of the cells, such as ALP activity, should be included in the cytotoxicity test of restorative dental materials.
In another test, the ranking of SDH activities for both HDPF and HPF were IRM = Dycal < FujiII = TimeLine = FujiIILC = Hy-Bond = Z100 < medium. It showed that all of the tested materials were toxic to the HDPF and HPF. But there were no significant difference of SDH activities between HDPF and HPF. The ranking of OD of ALP activities were Dycal < IRM = FujiIILC < TimeLine = FujiII < Hy-Bond = Z100 < medium. It showed that all of the tested materials inhibited ALP activities of HDPF and HPF. And there were significant difference of ALP activities between HDPF and HPF. The ALP activities of HDPF is significantly lower than those of HPF. It was suggested that material toxicities test for HPDF and HPF should be performed respectively. It also showed that the reparative dentin formation ability of deciduous teeth is lower than that of permanent teeth.
The third purpose of the study was to compare the content of fibronectin, collagen type I and collagen type III between deciduous pulpal fibroblasts (D) and permanent pupal fibroblasts (P) with 3T3 (T) and gingival fibroblasts (G) as control groups. The means were analyzed by MANOVA and then Duncan Test. It found that the OD ranking of fibronectin was T < D = P = G, the OD ranking of collagen type I was T < D = P < G, the OD ranking of collagen type III was T < P < D < G. According to these finding that fibronectin and collagen type I content of HDPF were equal to that of HPF, it concluded that HDPF preserved the productivities of proteins related to calcification as HPF. The contents of collagen type III of D and P cells were great than that of T cell and lesser than G cell, but had significant difference between D and P cells. According to the finding that the contents of collagen type III of HDPF were great than that of HPF, it concluded that the calcification ability of HDPF might be impaired.
Key words: Deciduous pupal fibroblast, cytotoxicity, alkaline phosphatase, succinate dehydrogenase, dental materials, capping materials, temporary filling materials, filling materials, fibronectin, collagen type I, collagen type III.
封面
1、中文摘要
2、英文摘要
3、致謝辭
4、 序言
5、材料與方法
6、 結果
7、 討論
8、 結論
9、 圖形與附表
10 、參考文獻
11、 附錄 1(儀器操作,藥品配製,測試步驟)
12、 附錄2 (論文投稿 JBMR 接受手稿)
13、附錄3 (論文投稿 23屆APDC 貼示報告)
14、 附錄4 (其他研究投稿 JCMS 發表)
15、 附錄5 (其他研究投稿 JCMS 接受)
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