In vitro transformation of periodontal ligament fibroblasts ( PF cells) and characterization

牙周病療法的精髓旨在確保遭牙周病破壞之組織得以再生。故此種再生首需針對牙周各附著器官進行重建工作，如新的骨形成、在暴露牙根表面沉積新的牙骨質（cementum）、以及將足以強化牙周韌帶功能的膠原 ( collegen ) 纖維插入新骨和牙骨質之內等。事實上，證據顯示職司形成此等組織的先軀細胞 ( progenitor cells )， 主要均源自牙周韌帶組織。因此，源自該處的細胞，想必在牙周再生的過程中，扮演關鍵的角色。基因或細胞療法是一個已然被各界認可的方法，因為它可把治療等級的蛋白質輸送到標的組織中，並能停留極長的一段時間。截至目前為止，活體外-「Ex vivo」基因療法在牙周齒槽骨缺損的修補方面，仍然是一項有待努力的挑戰，主因是利用較安全的非病毒載體（non-viral vectors）方法， 要把適當基因植入那些源自牙周韌帶組織之自源性 ( autologous ) 細胞內，並獲致極高的基因轉殖效率並不容易。雖然病毒載體（viral vectors）能以極高的效率轉型（transform）體外培養的細胞，並在諸多危及生命的情況下, 被視為用途極大的基因療法（gene therapy）工具，但在諸多不致危及生命情況下進行基因轉殖時， 採用非病毒載體的方法，仍然是一個亟欲追求的目標。例如，在修復各種牙周齒槽骨缺損方面，此等方法仍被視為一個非常有潛力的途徑。再者，腺病毒載體 (adenoviral vector ) 可能在某些情況下引發寄生主的免疫反應，而反轉錄病毒載體 (retroviral vector) 卻必須在細胞分裂的情況下，才能嵌合入細胞內。接著，還可能存在的一個不大的機率是，病毒載體可能在無意中被嵌合到寄主的染色體 （genome）內，促成轉變為腫瘤變性 (neoplastic transformation) 的危險。因此，尋求針對培養自源性細胞之高效率非病毒載體的基因轉殖方法，或能排除此種免疫反應的機會。由於基因療法已被認為是將來可能治療牙周齒槽骨缺損的重要方法之一，而針對牙周韌帶纖維母細胞 ( periodontal ligament fibroblasts, PF cells )，將外生性DNA（exogenous DNA）植入此種細胞的方法與可行性，研究很少而結果其實相當分岐，主要是缺乏有系統的分析。因此，我們評估並比較了數種非病毒性基因轉移的方法，包括磷酸鈣沉澱法（calcium phosphate-DNA precipitate method）、電破法（Electroporation）、LipofectamineTM 試劑和LipofectamineTM 2000 試劑，以作為有效將外生性DNA 送到培養的牙周韌帶纖維母細胞之可能工具。至於轉遞感染（transfection）條件的決定， 則利用「綠螢光蛋白質」( green fluorescent protein；GFP ) 作為報告基因，並搭配流式細胞儀 ( Flow cytometry ) 來定量轉遞感染的效率。我們獲致的結論是: 第一， 以LipofectamineTM 2000 試劑，並藉GFP 報告基因所進行的轉遞感染，其達成的效果，比較其他非病毒基因轉移法的效率要高出許多（高達66%）；第二，磷酸鈣沉澱法和電破法的轉遞感染的效率則不到10%；第三，LipofectamineTM 試劑的轉遞感染（transfection）效率，大約為25%。接著，我們進一步探討，外生性DNA是否能夠在PF細胞長久培養中持續表現？並以SV40 large T 和GFP 的質體 ( plasmid ) 來轉型PF 細胞，而轉型後的PF 細胞則利用RT-PCR的方法（mRNA等級）以及西方轉漬法（蛋白質等級）加以定性分析。經長期培養實驗證實，我們已成功獲致GFP and SV40 Large T 的永久轉遞感染牙周韌帶纖維母細胞株，它們能夠持續表現外生性DNA所encode的蛋白質至少三個月之久。 某些特定的骨生成標記，如第一型膠原蛋白 ( Type I collagen；Hco I )、鹼性磷酸酶 ( Alkaline phosphatase；ALPase )、骨鈣蛋白質 ( Osteocalcin；OCN ) 等，均能在培養的牙周韌帶纖維母細胞及SV40 Large T 的永久轉遞感染牙周韌帶纖維母細胞株中顯示出來。總之，LipofectamineTM 2000 試劑在培養的牙周韌帶纖維母細胞轉型方面, 已然顯示其相當不錯的效率，而且很可能在一些不危及生命的情況下， 成為一個非常實用的基因轉移的工具。至於SV40 Large T 的永久轉遞感染牙周韌帶纖維母細胞株，其已然較之原始的牙周韌帶纖維母細胞多繁殖了20代且並無太大的表現型 （phenotype）上的差異，因此，此經由LipofectamineTM 2000 試劑轉遞感染而來的細胞株不但有機會成為長生不老 ( immortalization ) 細胞，並在牙周再生的研究領域，可能成為牙周組織再生的細胞來源。The core of periodontal therapy is to ensure the regeneration of the tissue that has been destroyed by periodontitis. So the periodontal regeneration requires restitution of the periodontal attachment apparatus, i.e., new bone formation, new cementum deposition upon the denuded root surface, and insertion of functionally-oriented new collegen fibers targeting the periodontal ligament（PL）into the new bone and new cementum. The origins of progenitor cells responsible for the formation of these tissues have long been suggested to be mainly derived from PL tissue. Therefore, cells derived from there must play a pivotal role in the process of periodontal regeneration. Gene or cell therapy is a recognized methodology in delivering the therapeutic-level proteins to the targeted tissue for extended period of time. To date, ex-vivo gene therapy in the area of periodontal bone repair has remained a challenge, primarily due to the difficulty of achieving high-efficiency gene transfer into primary autologous cells derived from periodontal ligament tissue with non-viral techniques. Although viral vectors have been shown an efficacy in transforming cultured cells with high efficiency and have also been regarded as very useful gene therapy tools, the use of non-viral methods for gene delivery in non life-threatening situations remains a desirable goal. For instance, it is potentially considered as an effective method for repairing various periodontal bone defects. However, adenovirus vectors may induce host immune- response in some cases, while retroviral vectors demand dividing cells for integration of transfected gene. Moreover, there exists a chance that viral vectors may randomly be integrated into the host genome, posing a risk of neoplastic transformation. If such is the case, that the quest for a high-efficient non-viral gene transfer methods aiming at the primary autologous cells would preclude the possibility of an immune-response. The introduction of exogenous DNA into these periodontal ligament fibroblasts is controversially discussed, mainly due to the lack of systematic analysis. Therefore, in the present study we examined comparatively four non-viral gene transfer methods: calcium phorsphate reagent, electroporotion, LipofectamineTM reagent, and LipofectamineTM 2000 reagent, as potential tools for a efficient delivery of DNA to the culture of the PL fibroblasts (PF cells). The conditions of cell transfection were determined by using enhanced green fluorescent protein (GFP) as a reporter gene that expressed under the transcriptional control of the human cytomegalovirus promoter/enhancer. Quantitative evaluation of representative transfection experiments by flow cytometry revealed that out of four non-viral gene transfer methods tested, LipofectamineTM 2000 reagent allowed for a significantly higher transfection efficiency (up to 66﹪). Transfection efficiency with calcium phorsphate reagent or electroporotion was＜10％, and approximately 25％ of the PF cells were transfected by LipofectamineTM reagent. Transformation of the PF cells was made with plasmid encoded SV40 large T and GFP and the transformed PF cells were characterized by RT-PCR (mRNA level) and western blotting (protein level) techniques. By way of this effort, long-term persistent expression of the exogenous DNA in PF cells were monitored. In conclusion: firstly, transfection with GFP reporter gene by liposome technique (LipofectamineTM 2000 reagent) resulted in a higher efficiency (up to 66﹪) than other nonviral gene transfer methods. Secondly, by liposome method, we have successfully obtained two stable transfectants with GFP and SV40 large T, respectively. Both cell lines were able to express the exogenous DNA at least 3 months at the protein level. Thirdly, the results of RT-PCR showed that some osteoblast-specific markers, such as Type I collagen (Hcol), alkaline phosphatase (ALPase), osteocalcin (OCN) were able to be expressed both in cultured PF cells and the stable SV40 large T transfectants. The PF cells transfected with SV40 large T, which have propagated over 20 passages, seemed to have little change of its phenotypes. Thus, the immortalization-competent PF cells via lipofactamine 2000 reagent would provide a useful cell source for research of periodontal regeneration in vitro.中文摘要…………………………………………1 英文摘要…………………………………………………………………3 緒論………………………………………………………………………6 文獻回顧…………………………………………………………………7 實驗材料及方法……………………………………………………….37 一、初代培養細胞和細胞株………………………………………37 二、構築質體………………………………………………………….40 三、細胞轉遞感染的方法…………………………………………….41 四、光學顯微鏡：相位差顯微鏡與螢光顯微鏡…………………….47 五、流式細胞儀……………………………………………………….48 六、建立GFP and SV40 Large T 的永久轉遞感染牙周韌帶纖 維母細胞株…………………………………………………………….49 七、基因構築質體轉殖的證實……………………………………….49 八、SV40 Large T 的永久轉遞感染牙周韌帶纖維母細胞株的 定性……………………………………………………………………59 實驗結果………………………………………………………………67 討論……………………………………………………………………76 結論……………………………………………………………………93 參考文獻………………………………………………………………94 圖集說明………………………………………………………………108 圖集……………………………………………………………………114 表……………………………………………………………………..13