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研究生:陳韋弘
研究生(外文):Wei-Hong Chen
論文名稱:人體關節老化與發炎反應研究(I):人體關節軟骨細胞之不朽化與定性
論文名稱(外文):Human Joint Aging and Inflammatory Studies (I) :Human Articular Chondrocytes Immortalization and Characterization
指導教授:鄧文炳鄧文炳引用關係
指導教授(外文):Win-Ping Deng Ph.D.
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:生物醫學材料研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:72
中文關鍵詞:關節軟骨細胞間質細胞分化不朽化關節老化關節發炎
外文關鍵詞:articular cartilageextracellular matrixcell differentiationimmortalizationcartilage agingcartilage inflammation
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關節老化及發炎有普遍化及年輕化之趨勢,其最大之原因是關節軟骨細胞的病變;但目前尚無保持原良好特性之不朽化軟骨細胞株,可以用來進行關節老化及發炎的分子基因的研究。
本研究成功地利用retrovirus infection system將HPV-16 E6/E7 DNA 感染且不朽化人類關節軟骨細胞(immortalized human articular chondrocytes),並對此不朽化軟骨細胞株 (immortalized cell line)進行實驗分析及細胞定性,利用RT-PCR、Western blotting、Alcian Blue staining、三度立體空間培養(3 dimensional cultures)以及Immunohistology等方法,來觀察證明此不朽化軟骨細胞株與初代軟骨細胞之間的差異性,並將此細胞株並命名為hPi cells。
經由各項實驗分析證明,此不朽化軟骨細胞株(hPi cells)已可被成功地培養超過六十代,而且定性分析結果顯示,軟骨細胞最主要的分化marker : type II collagen,不論是在mRNA level或是Protein level,都可以在此hPi cells明顯偵測出和初代軟骨細胞有相同的表現,而且將hPi cells植入type I collagen為scaffold的三度空間立體培養中,在hPi cells的周圍亦有明顯的lacunae產生,經由對GAGs的專一性Alcian Blue染色結果,不論是初代軟骨細胞或是hPi cells,都可明顯被染色。由於此細胞株是經由HPV-16 E6/E7 DNA之轉感染,亦作了hPi cells在NOD/SCID mice身上是否會生成腫瘤(tumorigenic)之測試,經過長達三個月的培養,hPi cells在mice身上並無任何腫瘤生成反應,亦證明了此細胞株與正常細胞相同性質。
本實驗室所建立之軟骨細胞株hPi cells不需經過re-differentiation的過程,即可在體外培養到接近五十代,仍然有明顯的軟骨細胞分化markers表現,諸如type II collagen、Aggrecan,甚至是在3-dimension的培養環境下,都可看出此hPi 細胞株和初代軟骨細胞具有相同之分化能力及特性,是其他報告文獻中所欠缺的。希望藉由此hPi細胞株之成功建立,除了作為人體軟骨再生研究外,更重要是進而拓寬進行關節老化及發炎分子基因的研究領域!
Articular aging and inflammation has became a tendency of being popular and young. Pathological changes of articular cartilage chondrocytes are the main reason of these diseases. There is, however, still no well-established immortalized articular chondrocyte system to be used to study the molecular mechanism of articular aging and inflammation.
In this experiment, we have successfully established an immortalized human articular chondrocyte transfected with HPV 16 E6/E7 DNA by using retrovirus infection system and this cell line, named hPi, was analyzed and well characterized. RT-PCR, Western blotting, Alcian Blue staining, 3 dimensional cultures and immunohistology were used to demonstrate the characteristic similarity between human articular immortalized and primary chondrocytes.
The results showed that the major differentiation marker of articular chondrocytes, type II collagen in mRNA level or protein level in hPi cells were apparently identical with that in primary chondrocytes over 60 passages culture. Lacunae were formed between cells and collagen matrices obviously in hPi cells cultured in type I collagen scaffold in 3 dimension. The GAGs-specific Alcian Blue staining also demonstrated that both primary chondrocytes and hPi cells were stained in blueness. Tumor was not formed on NOD/ SCID mice injected with hPi cells for 3 months. This result indicated that the hPi cell line, chondrocytes immortalized by HPV-16 E6/E7, is non-tumorigenic and could still express cartilage-specific differentiation markers, such as type II collagen and aggrecan through 50 passages in vitro cultures without any redifferentiation strategies. The immortalized human articular chondrocytes will be used to investigate human articular regeneration, cartilage aging and inflammation molecular mechanisms !
目次
中文摘要…………………………………………………第i頁
英文摘要…………………………………………………第iii頁
圖表目錄…………………………………………………第viii頁
第壹章 緒論…………………………………………….第1頁
第貳章 研究材料與方法
第一節 初代培養………………………………….第9頁
第二節 HPV-16 E6/E7基因之轉感染……………第10頁
第三節 細胞增生速率………….…………………第11頁
第四節 total mRNA萃取.………………………..第12頁
第五節 反轉錄酵素-聚合酶連鎖反應…………...第13頁
第六節 細胞蛋白質萃取………………………….第15頁
第七節 蛋白質濃度測定………………………….第16頁
第八節 西方點墨法……………………………….第17頁
第九節 第一型膠原蛋白之純化萃取…………….第19頁
第十節 體外膠原蛋白三维立體空間培養……….第20頁
第十一節 體內膠原蛋白三维立體空間培養…….第21頁
第十二節 細胞蛋白質染劑染色……………...…..第22頁
第十三節 細胞腫瘤生成能力分析..…………….第23頁
第叁章 實驗結果與分析
第一節 細胞外觀型態……………………………..第24頁
第二節 HPV-16 E6/E7 mRNA於細胞內之偵測…第25頁
第三節 細胞於體外培養之基因表現偵測………..第26頁
第四節 細胞增生能力測試………………………..第28頁
第五節 西方點墨法..……………………………..第29頁
第六節 第一型膠原蛋白之純化…………………..第30頁
第七節 組織切片…………………………………..第31頁
第八節 細胞蛋白質染劑染色………………….….第33頁
第九節 腫瘤生成能力分析…………………….….第34頁
第肆章 討論………………………………………….….第35頁
圖與表……………………………………………….…….第43頁
參考文獻…………………………………………………..第59頁
附錄
附錄一、實驗設計………………………………………..第65頁
附錄二、WST-1作用示意圖………….……...…………..第66頁
附錄三、反轉錄酵素反應步驟…………………………..第67頁
附錄四、聚合酶連鎖反應………………………………..第68頁
附錄五、Bio-100TM DNA ladder..…….………………….第69頁
附錄六、SDS-PAGE緩衝液製備.…...…………………..第70頁
附錄七、SDS-PAGE電泳膠片製備.…………………….第71頁
附錄八、蛋白質電泳Maker,SeeBlue® Plus2…..…..….第72頁
1. Hunziker, E. B. (2002) Articular cartilage repair: basic science and clinical progress. A review of the current status and prospects. Osteoarthritis Cartilage 10, 432-463
2. Hardingham, T., Tew, S., Murdoch, A. (2002) Tissue engineering: chondrocytes and cartilage. Arthritis Res. 4 Suppl 3, S63-S68
3. Loeser, R. F., Sadiev, S., Tan, L., Goldring, M. B. (2000) Integrin expression by primary and immortalized human chondrocytes: evidence of a differential role for alpha1beta1 and alpha2beta1 integrins in mediating chondrocyte adhesion to types II and VI collagen. Osteoarthritis.Cartilage. 8, 96-105
4. van der Kraan, P. M., Buma, P., van Kuppevelt, T., van den Berg, W. B. (2002) Interaction of chondrocytes, extracellular matrix and growth factors: relevance for articular cartilage tissue engineering. Osteoarthritis Cartilage 10, 631-637
5. Karin Benz1, S. B. M. L. H. M. a. W. R. (2002) Molecular analysis of expansion, di.erentiation, and growth factor treatment of human chondrocytes identies dierentiation markers and growth-related genes. Biochemical and Biophysical Research Communications 293, 284-292
6. Temenoff, J. S., Mikos, A. G. (2000) Review: tissue engineering for regeneration of articular cartilage. Biomaterials 21, 431-440
7. M.Schnabel*, S. M. G. E. I. F. L. G. V. V. c. a. J. S. (20010) Dedifferentiation-associated changes in morphology and gene expression in primary human articular chondrocytes in cell culture. Osteoarthritis and Cartilage 10, 62-70
8. Thomas, B., Thirion, S., Humbert, L., Tan, L., Goldring, M. B., Bereziat, G., Berenbaum, F. (2002) Differentiation regulates interleukin-1beta-induced cyclo-oxygenase-2 in human articular chondrocytes: role of p38 mitogen-activated protein kinase. Biochem.J. 362, 367-373
9. Goldring, M. B., Birkhead, J. R., Suen, L. F., Yamin, R., Mizuno, S., Glowacki, J., Arbiser, J. L., Apperley, J. F. (1994) Interleukin-1 beta-modulated gene expression in immortalized human chondrocytes. J.Clin.Invest 94, 2307-2316
10. Goldring, M. B., Suen, L. F., Yamin, R., Lai, W. F. (1996) Regulation of Collagen Gene Expression by Prostaglandins and Interleukin-1beta in Cultured Chondrocytes and Fibroblasts. Am.J.Ther. 3, 9-16
11. Stokes, D. G., Liu, G., Coimbra, I. B., Piera-Velazquez, S., Crowl, R. M., Jimenez, S. A. (2002) Assessment of the gene expression profile of differentiated and dedifferentiated human fetal chondrocytes by microarray analysis. Arthritis Rheum. 46, 404-419
12. van Osch, G. J., van der Veen, S. W., Verwoerd-Verhoef, H. L. (2001) In vitro redifferentiation of culture-expanded rabbit and human auricular chondrocytes for cartilage reconstruction. Plast.Reconstr.Surg. 107, 433-440
13. Haudenschild, D. R., McPherson, J. M., Tubo, R., Binette, F. (2001) Differential expression of multiple genes during articular chondrocyte redifferentiation. Anat.Rec. 263, 91-98
14. Jakob, M., Demarteau, O., Schafer, D., Hintermann, B., Dick, W., Heberer, M., Martin, I. (2001) Specific growth factors during the expansion and redifferentiation of adult human articular chondrocytes enhance chondrogenesis and cartilaginous tissue formation in vitro. J.Cell Biochem. 81, 368-377
15. Pecina, M., Jelic, M., Martinovic, S., Haspl, M., Vukicevic, S. (2002) Articular cartilage repair: the role of bone morphogenetic proteins. Int.Orthop. 26, 131-136
16. Blunk, T., Sieminski, A. L., Gooch, K. J., Courter, D. L., Hollander, A. P., Nahir, A. M., Langer, R., Vunjak-Novakovic, G., Freed, L. E. (2002) Differential effects of growth factors on tissue-engineered cartilage. Tissue Eng 8, 73-84
17. Pei, M., Seidel, J., Vunjak-Novakovic, G., Freed, L. E. (2002) Growth factors for sequential cellular de- and re-differentiation in tissue engineering. Biochem.Biophys.Res.Commun. 294, 149-154
18. Kaps, C., Bramlage, C., Smolian, H., Haisch, A., Ungethum, U., Burmester, G. R., Sittinger, M., Gross, G., Haupl, T. (2002) Bone morphogenetic proteins promote cartilage differentiation and protect engineered artificial cartilage from fibroblast invasion and destruction. Arthritis Rheum. 46, 149-162
19. Hutmacher, D. W. (2000) Scaffolds in tissue engineering bone and cartilage. Biomaterials 21, 2529-2543
20. Rotter, N., Aigner, J., Naumann, A., Planck, H., Hammer, C., Burmester, G., Sittinger, M. (1998) Cartilage reconstruction in head and neck surgery: comparison of resorbable polymer scaffolds for tissue engineering of human septal cartilage. J.Biomed.Mater.Res. 42, 347-356
21. Saldanha, V., Grande, D. A. (2000) Extracellular matrix protein gene expression of bovine chondrocytes cultured on resorbable scaffolds. Biomaterials 21, 2427-2431
22. Grigolo, B., Roseti, L., Neri, S., Gobbi, P., Jensen, P., Major, E. O., Facchini, A. (2002) Human articular chondrocytes immortalized by HPV-16 E6 and E7 genes: Maintenance of differentiated phenotype under defined culture conditions. Osteoarthritis.Cartilage. 10, 879-889
23. Pieper, J. S., van der Kraan, P. M., Hafmans, T., Kamp, J., Buma, P., van Susante, J. L., van den Berg, W. B., Veerkamp, J. H., van Kuppevelt, T. H. (2002) Crosslinked type II collagen matrices: preparation, characterization, and potential for cartilage engineering. Biomaterials 23, 3183-3192
24. van Beuningen, H. M., Stoop, R., Buma, P., Takahashi, N., van der Kraan, P. M., van Den Berg, W. B. (2002) Phenotypic differences in murine chondrocyte cell lines derived from mature articular cartilage. Osteoarthritis.Cartilage. 10, 977-986
25. Alsberg, E., Anderson, K. W., Albeiruti, A., Rowley, J. A., Mooney, D. J. (2002) Engineering growing tissues. Proc.Natl.Acad.Sci.U.S.A 99, 12025-12030
26. Gruber, H. E., Hanley, E. N., Jr. (2000) Human disc cells in monolayer vs 3D culture: cell shape, division and matrix formation. BMC.Musculoskelet.Disord. 1, 1
27. Khoobyarian, N., Marczynska, B. (1993) Cell immortalization: the role of viral genes and carcinogens. Virus Res. 30, 113-128
28. Choo, C. K., Ling, M. T., Chan, K. W., Tsao, S. W., Zheng, Z., Zhang, D., Chan, L. C., Wong, Y. C. (1999) Immortalization of human prostate epithelial cells by HPV 16 E6/E7 open reading frames. Prostate 40, 150-158
29. Mataga, N., Tamura, M., Yanai, N., Shinomura, T., Kimata, K., Obinata, M., Noda, M. (1996) Establishment of a novel chondrocyte-like cell line derived from transgenic mice harboring the temperature-sensitive simian virus 40 large T-antigen gene. J.Bone Miner.Res. 11, 1646-1654
30. Chano, T., Okabe, H., Saeki, Y., Ishizawa, M., Matsumoto, K., Hukuda, S. (1998) Characterization of a newly established human chondrosarcoma cell line, CS-OKB. Virchows Arch. 432, 529-534
31. Takigawa, M., Tajima, K., Pan, H. O., Enomoto, M., Kinoshita, A., Suzuki, F., Takano, Y., Mori, Y. (1989) Establishment of a clonal human chondrosarcoma cell line with cartilage phenotypes. Cancer Res. 49, 3996-4002
32. Domm, C., Schunke, M., Christesen, K., Kurz, B. (2002) Redifferentiation of dedifferentiated bovine articular chondrocytes in alginate culture under low oxygen tension. Osteoarthritis.Cartilage. 10, 13-22
33. Kokenyesi, R., Tan, L., Robbins, J. R., Goldring, M. B. (2000) Proteoglycan production by immortalized human chondrocyte cell lines cultured under conditions that promote expression of the differentiated phenotype. Arch.Biochem.Biophys. 383, 79-90
34. Harms, W., Rothamel, T., Miller, K., Harste, G., Grassmann, M., Heim, A. (2001) Characterization of human myocardial fibroblasts immortalized by HPV16 E6--E7 genes. Exp.Cell Res. 268, 252-261
35. Takazawa, Y., Nifuji, A., Mataga, N., Yamauchi, Y., Kurosawa, H., Noda, M. (1999) Articular cartilage cells immortalized by a temperature sensitive mutant of SV40 large T antigen survive and form cartilage tissue in articular cartilage environment. J.Cell Biochem. 75, 338-345
36. Wambach, B. A., Cheung, H., Josephson, G. D. (2000) Cartilage tissue engineering using thyroid chondrocytes on a type I collagen matrix. Laryngoscope 110, 2008-2011
37. Ushida, T., Furukawa, K., Toita, K., Tateishi, T. (2002) Three-dimensional seeding of chondrocytes encapsulated in collagen gel into PLLA scaffolds. Cell Transplant. 11, 489-494
38. van Susante, J. L., Buma, P., van Osch, G. J., Versleyen, D., van der Kraan, P. M., van der Berg, W. B., Homminga, G. N. (1995) Culture of chondrocytes in alginate and collagen carrier gels. Acta Orthop.Scand. 66, 549-556
39. Schuman, L., Buma, P., Versleyen, D., de Man, B., van der Kraan, P. M., van den Berg, W. B., Homminga, G. N. (1995) Chondrocyte behaviour within different types of collagen gel in vitro. Biomaterials 16, 809-814
40. Kamil, S. H., Aminuddin, B. S., Bonassar, L. J., Silva, C. A., Weng, Y., Woda, M., Vacanti, C. A., Eavey, R. D., Vacanti, M. P. (2002) Tissue-engineered human auricular cartilage demonstrates euploidy by flow cytometry. Tissue Eng 8, 85-92
41. Kusuzaki, K., Sugimoto, S., Takeshita, H., Murata, H., Hashiguchi, S., Nozaki, T., Emoto, K., Ashihara, T., Hirasawa, Y. (2001) DNA cytofluorometric analysis of chondrocytes in human articular cartilages under normal aging or arthritic conditions. Osteoarthritis.Cartilage. 9, 664-670
42. 華人骨科專業資訊網 http://www.chinese-ortho.com/
43. ORTHOWORLD http://www.orthoworld.com/
44. American Academy of Orthopaedic Surgeons http://www.aaos.org/
45. Principle of Tissue Engineering Bobert. P.Lanza
Robert Langer Joseph Vacanti
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1. 林美和(民85,12月)。終身教育體系的學校圖書館定位與角色功能。高中圖書館館訊,17,18-22。
2. 林美和(民85,6月)。資訊素養與終身學習的關係。社教雙月刊,73,7-12。
3. 林美和(民64,10月)。從教育改革論學校圖書館計劃的革新。圖書館學與資訊科學,1(2),106-122。
4. 李德竹(民89,11月)。資訊素養的意義、內涵與演變。圖書與資訊學刊,35,1-25。
5. 李宗薇(民87,10月)。新世紀的衝擊與基礎教育的因應:談國小圖書館媒體中心的定位。研習資訊,15(5),35-47。
6. 吳茜茵(民84,2月)。國民小學圖書館媒體中心之經營。國立臺北師院圖書館館訊,3,21-29。
7. 林菁(民88,2月)。從資源本位學習到談學校教學資源中心之設立。研習資訊,16(1),36-48。
8. 林菁(民88,2月)。從資源本位學習到談學校教學資源中心之設立。研習資訊,16(1),36-48。
9. 林菁(民88,2月)。從資源本位學習到談學校教學資源中心之設立。研習資訊,16(1),36-48。
10. 張明輝(民88)。九O年代中小學學校教育革新之策略與展望。在國立臺灣師範大學教育學系編,教育研究集刊第43輯(頁103-137)。台北市:師大書苑。
11. 楊美華(民85,6月)。臺灣學術網路對學習中心的衝擊,高中圖書館,15,23-29。
12. 劉慶仁(民88,12月)。美國中小學教育改革的趨勢:從學童教育卓越法談起。文教新潮,4(4),3-7。
 
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