李伯訓Lee, Bor-Shiunn臺灣大學:臨床牙醫學研究所張懿丹Chang, Yi-TanYi-TanChang2010-05-262018-07-092010-05-262018-07-092008U0001-1306200801485900http://ntur.lib.ntu.edu.tw//handle/246246/184142牙齒常因牙周疾病、齲齒、撞傷或癌症而造成缺損，如果能發展出生物性的再生牙齒以取代缺損的牙齒將是臨床治療的一種新療法。本研究之目的是應用乳牙牙髓幹細胞，結合組織工程的方法達成牙髓組織的再生。首先，我們實驗利用酵素分解的方法，可以從人類脫落的乳牙分離出牙髓幹細胞，乳牙牙髓幹細胞能夠形成細胞群落(colony)，這顯示這些細胞有很好的增生及自我更新的能力，同時這些乳牙牙髓幹細胞會表現三種與幹性相關的轉錄因子: Oct4、Sox2和Nanog，以及多種幹細胞的表面標誌: STRO-1和CD146、CD29、CD44、CD90及CD105。在體外培養誘導下，乳牙牙髓幹細胞可以分化成骨母細胞、脂肪細胞或牙本質母細胞。在動物實驗中，我們將迷你豬乳牙的前牙作拔髓，後牙做斷髓處理，各分成四組做實驗，第一組不放任何細胞、第二組加入迷你豬乳牙牙髓幹細胞和培養液、第三組加入迷你豬乳牙牙髓幹細胞和lovastatin，第四組加入迷你豬乳牙牙髓幹細胞和odontoblast induction medium，觀察三個月後，以探討其牙齒組織是否會再生。結果發現會有再生性牙本質及collagen的產生。在後牙的組別，形成的再生性牙本質的量在各組並無差異；在前牙的組別，第三組加入lovastatin的牙齒，根管內產生了7.5 mm再生性牙本質，比第一組(4.0 mm)及第四組多(5.5 mm)，所以lovastatin可能會促進牙本質母細胞的分化。總括來說，本研究建立了一個新的研究牙髓幹細胞應用於牙齒再生治療的模式，我們發現從乳牙中可以分離出牙髓幹細胞，未來並可進一步應用來修復牙齒的缺損。Periodontal diseases、caries、trauma or cancer are common causes for tooth decay or tooth loss. A biological tooth substitute that could replace decayed tooth or lost tooth would provide a vital alternative to currently available clinical therapy. The aim of the study was to utilize deciduous dental pulp stem cells and tissue engineering method to regenerate injured dental pulp. By using enzyme digestion, dental pulp stem cells could be isolated from deciduous teeth. These cells have capacity to generate clongenic cell clusters in culture. They were also identified to be a population with high proliferation capability. They can express three stemness genes- Oct4, Sox2 and Nanog and surface markers which are known to express in stem cells, such as STRO-1, CD146, CD29, CD44, CD90 and CD105 were also expressed on these cells. Moreover, these deciduous dental pulp stem cells could differentiate into osteoblasts, adipocytes, or odontoblast. We also established an animal model with utilizing mini-pig to determine the functional property of dental pulp stem cell in vivo. Firstly, we performed pulpotomy for posterior teeth and pulpectomy for anterior teeth. And the experiments were divided into four groups. In the first group, nothing was put in the pulp chamber or canals. In the second group, deciduous dental pulp stem cells of mini-pig with cultured medium were transplanted into the pulp chamber and pulp canals. In the third group, deciduous dental pulp stem cells of mini-pig with lovastatin were transplanted. In the fourth group, deciduous dental pulp stem cells of mini-pig with odontoblast induction medium were transplanted. Then we determined the pulp reaction three month after transplantation. The result showed that reparative dentin and collagen were regenerated. The amount of regenerative dentin in each group of posterior teeth was roughly similar. However, in the third group of anterior teeth, in which lovastatin was added, 7.5 mm reparative dentin was regenerated. The regenerated amount was greater than those in the first and the fourth groups. Therefore, lovastatin might enhance odontoblast differentiation. In summary, we established a new model for investigating the functional reconstruction capability with dental pulp stem cells transplantation in vivo. We also revealed dental pulp stem cells from one deciduous tooth can be expanded in vitro which possibly can be utilized for teeth replacement therapy in the future.中英文名詞及縮寫對照表---------------------------------------------------------------------I次---------------------------------------------------------------------------------------------VII次----------------------------------------------------------------------------------------------IX文摘要-----------------------------------------------------------------------------------------X bstract-----------------------------------------------------------------------------------------XII一章 文獻回顧-------------------------------------------------------------------------------1一節前言--------------------------------------------------------------------------------1二節 牙齒的發育----------------------------------------------------------------------1三節 牙髓的組織及功能-------------------------------------------------------------3四節 牙髓幹細胞----------------------------------------------------------------------5.1牙髓幹細胞在體內的最佳處所（Perivascular niche of pulp stem cell)-6.2 牙髓幹細胞與骨髓幹細胞之比較--------------------------------------------8.3 骨髓間質幹細胞與牙髓幹細胞織表面抗原標幟比較--------------------8五節 牙齒組織工程-------------------------------------------------------------------9.1牙齒組織工程過去研究成果--------------------------------------------------11.2 牙齒組織工程現況-------------------------------------------------------------12二章 研究材料與方法-------------------------------------------------------------------17一節 所使用細胞株及細胞培養條件--------------------------------------------17.1 培養牙髓幹細胞之相關藥品配製-------------------------------------------17.1.1 牙髓幹細胞培養液之配製----------------------------------------------18.1.2 磷酸鹽緩衝溶液(Phosphate buffered solution；PBS)配製--------18.1.3 1倍之0.25%胰蛋白酵素(Trypsin)-0.02﹪乙烯二胺酸(Ethylene diamietrtraacetic acid；EDTA)配製-------------------------------------------18二節 人類乳牙牙髓幹細胞之培養-----------------------------------------------18.1. 人類乳牙牙髓組織之取得---------------------------------------------------19.2. 乳牙牙髓幹細胞之培養------------------------------------------------------19.3. Colony forming assay ---------------------------------------------------------20.4 分裂複製能力試驗結果-BrdU assay (proliferation activity)------------21.5. 人類乳牙牙髓幹細胞進行流式細胞儀分析(Flow cytometry)--------22.6. 免疫組織化學染色(Immunohistochemical staining)--------------------22三節 人類乳牙牙髓幹細胞分化能力實驗--------------------------------------23.1. 骨母細胞誘導分化(Osteoblast differentiation)---------------------------23.1.1. 骨母細胞誘導分化培養液之配製(Osteoblast induction medium)-----------------------------------------------------------------------------23.1.2. 骨母細胞誘導分化(Osteoblast differentiation)---------------------23.1.3. Alizarin Red 染色 ---------------------------------------------------23.1.4. ALP stain鹼性磷酸酶染色----------------------------------------------243.2. 脂肪細胞誘導分化(Adipocyte Differentiation)-------------------------25.2.1. 脂肪細胞誘導分化培養液之配製(Adipocyte induction medium)- -----------------------------------------------------------------25.2.2. 脂肪細胞誘導分化(Adipocyte Differentiation) --------------------25.2.3. Oil Red-O染色------------------------------------------------------------26.2.3.1 Oil Red-O染劑的製備----------------------------------------------26.2.3.2 Oil Red-O染色操作過程-------------------------------------------26.3.牙本質母細胞誘導分化(Odontoblast Differentiation)-------------------26四節 反轉錄聚合酵素鏈鎖反應（PCR) -----------------------------------------27.1. Total RNA之萃取--------------------------------------------------------------27.2. RNA定量------------------------------------------------------------------------28.3. RNA反轉錄成cDNA (Reverse Transcription) ---------------------------28.4. 反轉錄聚合酶反應(Reverse transcription-polymeraes chain reaction)- ------------------------------------------------------------------------------------------28.5. 1% Agarose gel的配置--------------------------------------------------------30.6. 電泳跑膠------------------------------------------------------------------------31五節 動物實驗-----------------------------------------------------------------------31.1. 實驗動物來源及飼養管理---------------------------------------------------31.2. 豬之手術及麻醉---------------------------------------------------------------31.3. 迷你豬乳牙牙髓幹細胞之培養---------------------------------------------32.4. 迷你豬乳牙牙髓幹細胞再生實驗------------------------------------------33.4.1.實驗豬隻牙齒組織學分析-----------------------------------------------34三章 實驗結果------------------------------------------------------------------------------35一節 人類乳牙牙髓幹細胞之培養------------------------------------------------35.1. Colony forming assay----------------------------------------------------------35.2. 分裂複製能力試驗結果- BrdU assay (proliferation activity) ---------35.3. 人類乳牙牙髓幹細胞進行流式細胞儀分析結果 (Flow cytometry)-36.4. 免疫組織化學染色結果 (Immunohistochemical staining)------------36.4.1. 人類乳牙牙髓幹細胞 (SHED) 幹性相關基因細胞免疫染色實驗結果--------------------------------------------------------------------------------36.4.2. 人類乳牙牙髓幹細胞 (SHED) 表面抗原免疫染色實驗結果-36.5人類乳牙牙髓幹細胞幹性相關基因PCR實驗結果-------------------37二節 人類乳牙牙髓幹細胞之分化能力實驗-----------------------------------37.1. 骨母細胞誘導分化結果(Osteoblast differentiation) --------------------37.2. 脂肪細胞誘導分化結果(Adipocyte Differentiation) --------------------37 2.3. 牙本質母細胞誘導分化結果(Odontoblast Differentiation) ------------38三節 動物實驗------------------------------------------------------------------------38.1. 豬牙髓幹細胞之取得及培養------------------------------------------------38.1.2 分裂複製能力試驗結果-BrdU assay----------------------------------38.2. 迷你豬株乳牙牙髓幹細胞移植實驗切片組織化學染色結果---------39四章 討論------------------------------------------------------------------------------------41一節人類乳牙牙髓幹細胞之培養-------------------------------------------------41二節人類乳牙牙髓幹細胞幹性相關基因----------------------------------------45三節人類乳牙牙髓幹細胞之間質幹細胞表面標幟----------------------------48四節牙髓幹細胞的來源-------------------------------------------------------------49五節人類乳牙牙髓幹細胞之分化能力-------------------------------------------52.1.骨母細胞誘導分化-------------------------------------------------------------52.2. 脂肪細胞誘導分化-----------------------------------------------------------54.3.牙本質母細胞誘導分化-------------------------------------------------------55六節 動物實驗-----------------------------------------------------------------------57五章 未來展望------------------------------------------------------------------------------63考文獻----------------------------------------------------------------------------------------64application/pdf2912772 bytesapplication/pdfen-US牙髓幹細胞乳牙間質幹細胞組織工程牙齒再生牙本質dental pulp stem celldeciduous toothmesenchymal stem celltissue engineeringtooth regenerationdentin[SDGs]SDG3http://ntur.lib.ntu.edu.tw/bitstream/246246/184142/1/ntu-97-R94422006-1.pdf