臺灣博碩士論文加值系統

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……………………………………………………………………..138

Alam, J., and L., c. J. (1990) Anal. Biochem. 188, 245–254.
Alexander N. Gubin, Bindu Reddy, Joyce M. Njoroge, and Jeffery L. Miller. 1997. Long-Term, Stable Expression of Green Fluorescent Protein in Mammalian Cells. Biochemical And Biophysical Research Communications 236, 347–350.
Andreas Lundqvist, Gabriele Noffz, Maxim Pavlenko, Stein Sæbøe-Larssen, Timothy Fong, Norman Maitland, and Pavel Pisa. 2002. Nonviral and Viral Gene Transfer Into Different Subsets of Human Dendritic Cells Yield Comparable Efficiency of Transfection . Journal of Immunotherapy 25(6): 445–454.
Baltzer1 A. and Lieberman JR. 2004. Regional gene therapy to enhance bone repair. Gene Therapy 11, 344–350.
Berger, J., Hauber, J., Hauber, R., Gieger, R., and Cullen, B. R. (1988) Gene 66, 1–10.
Blott EJ and Griffiths GM. 2002. Secretory Lysosomes. Nature Reviews, Molecular Cell Biology 3, 122–131.
Bronstein, I., Fortin, J., Stanley, P. E., Stewart, G. S., and Kricka, L. J. (1994) Anal. Biochem. 219, 169 –181.
Canalis E, McCarthy TL, and Centrella M. 1989. Effects of platelet-derived growth factor on bone formation in vitro. J Cell Physiol 140:530-537.
Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., and Prasher, D. C. 1994．Science 263, 802–805.
Chang SC-N, Chuang HL, Chen YR, Chen JK, Chung H-Y , Lu Y-L , Lin H-Y , Tai C-L and Lou J . 2003. Ex vivo gene therapy in autologous bone marrow stromal stem cells for tissue-engineered maxillofacial bone regeneration. Gene Therapy, 10, 2013–2019.
Chen Y, Cheung K, Kung H, Leong J, Lu W, and Luk K. 2002. In vivo new bone formation by direct transfer of adenoviral-mediated bone morphogenetic protein-4 gene. Biochemical and Biophysical Research Communications, 298, 121–127.
Colman K. Byrnes B., Petra H., Mark D. and John W. H. 2002. A Nuclear Targeting Peptide Improves Transfection Efficiency in Fibroblasts. Journal of Surgical Research , 108, 85–90
Coonrod A, Li F-Q, and Horwitz M. 1997.On the mechanism of DNA transfection: efficient gene transfer without viruses. Gene Therapy , 4, 1313–1321
Crisco J, Jolk P, and Heinen G. 1994. A muscle contusion injury model, biomechanics, physiology and histology. Am J Sports Med , 22, 702–710.
de Wet, J. R., Wood, K. V., DeLuca, M., Helinski, D. R., and Subramani, S. 1987. Mol. Cell. Biol. 7, 725–737.
Doh SG, Vahlsing HL, Hartikka J, Liang X, and Manthorpe M. 1997. Spatial-temporal patterns of gene expression in mouse skeletal muscle after injection of IacZ plasmid DNA. Gene Therapy, 4, 648-663.
Einhorn TA. 2003. Clinical Applications of Recombinant Human BMPs: Early Experience and Future Development. The Journal Of Bone & Joint Surgery,85-A · Supplement 3 ,82-88.
Fanning E and knippers R.1992.Structureand function of simian virus 40 large tumor antige.Ann.Rev.Biochem., 61, 55-85.
Felgner, P. L., and Ringold, G. M. (1989) Nature 337, 387–388.
Fromigue O, Louis K, Dayem M, Milanini J, Pages G, Deckert ST, Ponzio P, Hofman P, Barbry P, Auberger P and Bernard Mari. 2003. Gene expression profiling of normal human pulmonary fibroblasts following coculture with non-small-cell lung cancer cells reveals alterations related to matrix degradation, angiogenesis, cell growth and survival. Oncogene , 22, 8487–8497.
Giannobile WV, Hernandez RA, Finkelman RD, Ryan S, Kiritsy CP, D’Andrea M, Lynch SE. 1996. Comparative effects of platelet-derived growth factor-BB and insulin-like growth factor-I, individually and in combination, on periodontal regeneration in Macaca fascicularis. J Periodontal Res, 31, 301-312.
Golzio Muriel, Mora MP, Raynaud C, Delteil C, Teissie´J, and Rols MP. Control by Osmotic Pressure of Voltage-Induced Permeabilization and Gene Transfer in Mammalian Cells. Biophysical Journal Volume 74 June 1998 3015–3022.
Goomer R S, Deftos LJ, Terkeltaub R, Maris T, Lee MC, Harwood FL and Amiel D. High-efficiency non-viral transfection of primary chondrocytes and perichondrial cells for ex-vivo gene therapy to repair articular cartilage defects. Osteoarthritis and Cartilage (2001) 9, 248–256.
Gorman, C. M., Moffat, L. G., and Howard, B. H. (1982) Mol. Cell. Biol. 2, 1044–1051.
Gysin R, Wergedal JE, Sheng MH-C, Kasukawa Y, Miyakoshi N, Chen S-T, Peng H, Lau K-HW, Mohan S and Baylink DJ. 2002. Ex vivo gene therapy with stromal cells transduced with a retroviral vector containing the BMP4 gene completely heals critical size calvarial defect in rats. Gene Therapy, 9, 991–999.
H. Schliephake. Bone growth factors in maxillofacial skeletal reconstruction. Int. J. Oral Maxillofac. Surg. 2002; 31: 469–484
Hammermeister KE, Sethi GK, Henderson WG, Oprian C, Kim T, Rahimtoola S. A comparison of outcomes in men 11 years after heart-valve replacement with a mechanical valve or bioprosthesis: Veterans Affairs Cooperative Study on Valvular Heart Disease. N Engl J Med. 1993;328:1289–1296.
Hoang A.M., Chen D., Oates T.W., Jiang C., Harris S.E., and Cochran D.L. 1997. Development and Characterization of a Transformed Human Periodontal Ligament Cell Line. J Periodontal; 68:1054–1062.
Hock JM, Centrella M, and Canalis E. 1988. Insulin-like growth factor I has independent effects on bone matrix formation and cell replication. Endocrinology, 122（1）:254-260,
Hou L. T. and Yaeger J. A. 1993. Cloning and Characterization of Human Gingival and Periodontal Fibroblasts. J Periodontal, 64, 1209–1218.
Hou L. T., Liu C. M., Lie J. Y., Wong M. Y. and Chen J. K. 2000. Biological Effects of Cementum and Bone Extracts on Human Periodontal Fibroblast. J Periodontal, 71, 1100–1109.
Howell TH, Fiorellini JP, Paquette DW, Offenbacher S, Giannobile WV, Lynvh SE. 1997. A phase I/II clinical trial to evaluate a combination of recombinant human platelet-derived growth factor-BB and recombinant human insulin-like growth factor-I in patient with periodontal disease. J Periodontal, 68（12）, 1186-1193.
Huard J, Yokoyama T, Pruchnic R, Qu Z, Li Y, Lee JY, Somogyi GT, Groat WC and Chancellor MB. 2002. Muscle-derived cell-mediated ex vivo gene therapy for urological dysfunction. Gene Therapy, 9, 1617–1626.
Isaka J., Ohazama A., Kobayashi M., Nagashima C., Takiguchi T.,Hideyuki H., Tachikawa T., and Hasegawa K. 2001. Participation of Periodontal Ligament Cells With Regrneration of Alveolar Bone. J Periodontal, 72, 314-323.
Ivanovski, Haase H.R., and Bartold P.M. 2001. Expression Of Bone Matrix Protein mRNAs by Primary and Cloned Cultures of the Regenerative Phenotype of Human Periodontal Fibroblasts. J Dent Res, 80(7), 1665-1671.
Iwaguro H, Yamaguchi J, Kalka C, Murasawa S, Masuda H, Hayashi S, Silver M, Li T, Isner JM, and Asahara T. 2002. Endothelial progenitor cell vascular endothelial growth factor gene transfer for vascular regeneration. Circulation, 105:732–738.
Jefferson, R. A., Kavanagh, T. A., and Bevan, M. W. (1987) EMBO J. 6, 3901–3907.
Jeffrey B. 2000. Tissue engineering via local gene delivery: Update and future prospects for enhancing the technology. Advanced Drug Delivery Reviews, 44, 185–194.
Jin Q, Anusaksathien O, Webb SA, Printz M, and Giannobile WV. 2004. Engineering of Tooth-Supporting Structures by Delivery of PDGF Gene Therapy Vectors. Molecular Therapy , 9, 519-526.
Jin Q-M.,Anusaksathien O., Webb S.A.,Rutherford R.B., and Giannobile W.V. 2003. Gene Therapy of Bone Morphogenetic Protein for Periodontal Tissue Engineering. J Periodontal, 74, 202-213.
Kafienah W, Al-Fayez F, Hollander AP, and Barker M. 2003. A Combined Tissue Engineering and Gene Therapy Approach. Arthritis & Rheumatism , 48, 709–718.
Kain, S. R. in Methods of Molecular Biology Series: Expression and Detection of Recombinant Genes (Tuan,R. S., Ed.), Humana Press, New Jersey, in press.
Kain, S. R., and Ganguly, S. 1995. in Current Protocols in Molecular Biology, John Wiley & Sons, New York.
Kamiya H, Yamazaki J and Harashima H. 2002.Size and topology of exogenous DNA as determinant factors of transgene transcription in mammalian cells. Gene Therapy, 9, 1500–1507.
Kasemkijwattana C, Menetrey J, and Somogyi G . 1998. Development of approaches to improve the healing following muscle contusion. Cell Transplant, 7, 585–598.
Kichler A, Zauner W, Ogris M and Wagner E. 1998. Influence of the DNA complexation medium on the transfection efficiency of lipospermine/DNA particles. Gene Therapy 5, 855–860.
Kramer B.P., Weber W., Fussenegger M. 2003. Artificial Regulatory Networks and Cascades for Discrete Multilevel Transgene Control in Mammalian Cells. Biotechnology And Bioengineering, 83:810–820.
Krebsbach PH, GU K, Franceschi RT, and Rutherford RB. 2000.Gene Therapy-Directed Osteogenesis: BMP-7-Transduced Human Fibroblasts Form Bone in Vivo. Human Gene Therpy, 11, 1201- 1210 .
Kuru L., Parkar M.H., Griffiths G.S., Newman H.N., and Olsen I. 1998. Flow Cytometry Analysis Gingival And Periodontal Ligament Cells. J Dent Res 77(4): 555-564.
Lanza R. P., Textbook：Principle of Tissue Engineering
Lieberman JR, Ghivizzani SC, and Evans CH. 2002. Gene Transfer Approaches to the Healing of Bone and Cartilage. Molecular Therapy, 6, 36-45.
Liechty KW, Mackenzie TC, Shaaban AF, Radu A. Human mesenchymal stem cells engraft and demonstrate sites pecific differentiation after in utero transplantation in sheep. Nature Medicine, 6, 1282-1286.
Liu H, Jan MS, Chou CK, Chen PH, and Ke NJ. 1999. Is Green Fluorescent Protein Toxic to the Living Cells? Biochemical and Biophysical Research Communications, 260, 712–717.
Luk K, Chen Y, Cheung K, Kung HF, Lu W, and Leong J. 2003. Adeno-associated virus-mediated bone morphogenetic protein-4 gene therapy for in vivo bone formation. Biochemical and Biophysical Research Communications, 308, 636–645.
Lynch SE, Castilla GR, Kiritsy CP, Howell TH, Reddy MS, Antoniades HN.1993. The effects of short-term application of a combinant of platelet-derived and insulin-like growth factors on periodontal wound healing. J Periodontal, 62（7）,458-467.
Manfredi JJ and Prives C. 1994. The transforming activity of simiun virus 40 large tumor antigen. BBA 1198:65-83.
Manfredi JJ and Prives C. 1994. The transforming activity of simiun virus 40 large tumor antigen. Bichim. Intern.,27:501-510
Matsuda N, Lin WL, Kumar NM, Cho MI, and Genco RJ. 1992. Mitogenic, chemotactic, and synthetic response of rat periodontal ligament fibroblastic cells to polypeptide growth factors in vitro.J Periodontol, 63（6）, 515-525.
Mayne LV, Priestly A, James MR, and Burke JF. 1986. Efficient Immortalization and Morphological Transformation of Human Fibroblasts by Transfection with SV40 DNA Linked to a Dominant Maker. Experimental cell research, 162, 530-538.
McCarthy TL, Centrella M, and Canalis E. 1989. Regulatory effects of insulin-like growth factors I and II on bone collagen synthesis in rat calvarial cultures. Endocrinology, 124（1）, 301-309.
McAuslan BR, and Johnson G. 1987. Cell responses to biomaterials, I: adhesion and growth of vascular endothelial cells on poly (hydroxyethyl methacrylate) following surface modification by hydrolytic etching. J Biomed Mater Res., 21, 921–935.
Migliaccio A.R., Bengra C., Ling J., Pi J., Li C., Zeng C., Keskintepe M., Whitney , Massimo S., Migliaccio G., Tuan D. 2000. Stable and unstable transgene integration sites in the human genome: extinction of the Green Fluorescent Protein transgene in K562 cells. Gene 256：197–214.
Moorwood K, Price T.N.C, And Mayne LV. 1996. Mutation of p53 Is Not a Prerequisite for Immortalization of Human Fibroblasts by SV40 T Antigen. Experimental Cell Research, 223, 308-313.
Moran E. 1988. A region of SV40 large T antigen can substitute for a transforming domain of the adenovirus E1A products. Nature, 334, 168- 170.
Mortimer I, Tam P, MacLachlan I, Graham RW, Saravolac EG and Joshi PB. 1999.Cationic lipid-mediated transfection of cells in culture requires mitotic activity. Gene Therapy, 6, 403–411.
Mu¨ller KJ, Horbaschek M, Lucas K, Zimmermann U, and Sukhorukova VL. 2003. Electrotransfection of anchorage-dependent mammalian cells. Experimental Cell Research , 288 , 344–353.
Nakashima M. and Reddi A. R. 2003. The application of bone morphogenetic proteins to dental tissue engineering. Nature biotechnology, 21, 1025-1032.
Nugent H, Edelman E. 2003. Tissue Engineering Therapy for Cardiovascular Disease. Circ Res., 92, 1068-1078.
Oates TW, Rouse CA, and Cochran DL. 1993. Mitogenic effects of growth factors on human periodontal ligament cells in vitro. J Periodontal, 64（2）, 142-148.
Park JB, Matsuda M, Han KY, Norderyd O, Lin WL, Genco RJ, and Cho MI. 1995. Periodontal regeneration in class III furcation defects of beagle dogs using guided tissue regenerative therapy with platelet-derived growth factor. J Periodontal, 66（6）, 462-477.
Parkara M.H., Kurua L., O''Harec M., Newmana H.N., Hughesd F., Olsena I., 1999. Retroviral transduction of human periodontal cells with a temperature-sensitive SV40 large T antigen. 44: 823–834.
Pfeilschifter J, Oechsner M, Naumann A, Gronwald RGK, Minne HW, and Ziegler R. 1990. Stimulationof bone matrix apposition in vitro by local growth factors: a comparison between insulin-like growth factor I, platelet-derived frowth factor, and transforming growth factor β. Endocrinology, 127（1）, 69-75.
Raouf A and Seth A 2000. Ets transcription factors and targets in osteogenesis. Oncogene 19, 6455 – 6463.
Redlich M, Roos H, Reichenberg E, Zaks B, Grosskop A, Bar Kana I, Pitaru S , and Palmon A. 2004.The effect of centrifugal force on mRNA levels of collagenase, collagen type-1, tissue inhibitors of metalloproteinases and β–actin cultured human periodontal ligament fibroblasts. J Periodontal Res, 39, 27- 32.
Sakurai F, Inoue R, Nishino Y, Okuda A, Matsumoto O, Taga T, Yamashita F, Takakura Y, and Hashida M. 2000. Effect of DNA/liposome mixing ratio on the physicochemical characteristics, cellular uptake and intracellular trafficking of plasmid DNA/ cationic liposome complexes and subsequent gene expression. Journal of Controlled Release, 66, 255–269.
Schliephake H. 2002. Bone growth factors in maxillofacial skeletal reconstruction. Int. J. Oral Maxillofac. Surg. , 31, 469–484.
Shay JW, Brigitte A. Van Der Haegen, Ying Y, and Woodring E. Wright. 1993.The Frequency of Immortalization of Human Fibroblasts and Mammary Epithelial Cells Transfected with SV40 Large T-Antigen. Experimental Cell Research , 209, 45-52 .
Shay JW, Woodring E. Wright and Harold Werbin. 1991. Defining the molecular mechanisms of human cell immortalization. Biochimica et biophysica acta, 1072, 1-7.
Springer M, Chen A, and Kraft P. 1998.VEGF gene delivery to muscle: potential role for vasculogenesis in adults. Mol Cell, 2, 549–558.
Takashiba S, Naruishi K, and Murayama Y.2003. Perspective Cytokine Regulation For Periodontal Treatment: Fibroblast Biology. J Periodontal , 8, 103-110.
Thomson TS, Berry JE, Somerman MJ and Kirkwood KL. 2003. Cementoblasts Maintain Expression of Osteocalcin in the Presence of Mineral Trioxide Aggregate. Journal Of Endodontics, 29, 407-412.
Uchida E, Mizuguchi H, Akiko I, and Takao H. 2002.Comparison of the Efficiency and Safety of Non-viral vector-Mediated Gene Transfer into a Wide Range of Human Cells. Biol. Pharm. Bull. , 25(7), 891—897.
Ulmer JJ. 1992.Heterologous protection against influenza by injection of DNA encoding a viral protein. Science, 259, 1745-1749.
Van der Kraan P, van de Loo F, van den Berg W. 2004. Role of gene therapy in tissue engineering procedures in rheumatology: the use of animal models. Biomaterials, 25, 1497–1504.
Wang Y, Rutherford B, Upholt WB and Mina M. 1999. Effects of BMP-7 on Mouse Tooth Mesenchyme and Chick Mandibular Mesenchyme. Developmental Dynamics, 216, 320-335.
Weiskirchen R, Kneifel J, Weiskirchen S, Eddy van de Leur, Kunz D and Gressner AM. 2000. Comparative evaluation of gene delivery devices in primary cultures of rat hepatic stellate cells and rat myofibroblasts. BMC Cell Biology, 1:4, 1471-1480.
Wheeler JJ, Palmer L, Ossanlou M, MacLachlan I, Graham RW, Zhang YP, Hope MJ, Scherrer P and Cullis PR. 1999. Stabilized plasmid-lipid particles: construction and Characterization. Gene Therapy, 6, 271–281.
Wolff JA et al. 1992. Long-term persistence of plasmid DNA and foreign gene expression in mouse muscle. Hum Mol Genet, 1, 363-369.
Wright VJ, Peng H, Usas A, Young B, Gearhart B, Cummins J, and Huard J. 2002. BMP4-Expressing Muscle-Derived Stem Cells Differentiate into Osteogenic Lineage and Improve Bone Healing in Immunocompetent Mice. Molecular Therapy, 6, 169-178.
Wu D, Razzano P, and Daniel A. 2003. Grande. Gene Therapy and Tissue Engineering in Repair of the Musculoskeletal System. Journal of Cellular Biochemistry, 88, 467–481.
Zhang G, Gurtu V, and Kain SR. 1996. An Enhanced Green Fluorescent Protein Allows Sensitive Detection of Gene Transfer in Mammalian Cells. Biochemical And Biophysical Research Communications , 227, 707–711.
Zhang X, Mao Z, and Yu C. 2004. Suppression of early experimental osteoarthritis by gene transfer of interleukin-1 receptor antagonist and interleukin-10. J Orthop Res., 22(4), 742-50.
Zhang Z, Song Y, Zhang X, Tang J, Chen J, Chen Y. 2003.Msx1/Bmp4 genetic pathway regulates mammalian alveolar bone formation via induction of Dlx5 and Cbfa1. Mechanisms of Development, 120 , 1469–1479.
Zhu Z., Lee C.S., Tejeda K.M., and Giannobile W.V. 2001. Gene Transfer and Expression of Platelet-derived Growth Factors Modulate Periodontal Cellular Activity. J Dent Res 80(3), 892-897.
Zuhorn I.S. and Hoekstra D., 2002. On the Mechanism of Amphiphile-mediated Transfection. To Fuse or not to Fuse : Is that the Question? J. Membrane Bio. 189, 167-179.