跳到主要內容

臺灣博碩士論文加值系統

(35.175.191.36) 您好!臺灣時間:2021/08/01 00:22
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:黃緯華
研究生(外文):Wei-Hua, Huang
論文名稱:建立使用骨髓幹細胞之骨塑型蛋白二慢病毒體外基因治療系統
論文名稱(外文):Establishment of a bone marrow stem cell- mediated BMP2 lentiviral ex vivo gene therapy system
指導教授:陳恆理陳恆理引用關係
指導教授(外文):Hen-Li Chen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:口腔生物研究所
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:2009
畢業學年度:97
語文別:中文
論文頁數:91
中文關鍵詞:骨塑型蛋白二基因治療異位骨生成慢病毒載體
外文關鍵詞:bone morphogenetic proteins 2BMP2gene therapyectopic bone formationlentiviral vector
相關次數:
  • 被引用被引用:0
  • 點閱點閱:122
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
臨床上常以植入骨移植物的方式治療骨缺損,但目前骨移植療法仍有不少限制,且在治療大型骨缺損時效果常未臻理想。組織工程使用支架、細胞與生長因子,為深具潛力之新興再生技術,應用組織工程於修復大型骨缺損時,支架需有足夠機械強度以維持再生所需空間,細胞宜採用具高度再生能力之幹細胞,而基因治療(gene therapy)可被應用於持續給予促進組織再生的因子,如此組合才易獲致最佳療效。本研究室先前已發展出聚乳酸-甘醇酸(poly (lactide-co-glycolide), PLGA)加上膠原蛋白(collagen)的具良好機械強度的複合支架。近年研究顯示同種異體骨髓幹細胞移植似乎不會產生免疫與排斥反應,且其組織修復及再生效果與使用自體骨髓幹細胞無異。骨塑型蛋白二(bone morphogenetic proteins 2, BMP2)為強效骨生長因子,利用慢病毒基因治療法則可長期表達因子。為將來以狗模型測試以組織工程策略治療大型骨缺損之效果,乃進行此研究以建立治療所需之組合性組織工程治療系統。本研究製備PLGA/collagen複合支架;分離具幹細胞特性的狗骨髓幹細胞;建構具促進骨分化作用的人類BMP2基因重組慢病毒載體(lentiviral vector);最後以嚴重免疫不全小鼠皮下異位骨生成模型證實所建立使用骨髓幹細胞之體外慢病毒BMP2基因治療系統具促進骨生成效果。本研究首次使用PLGA/collagen複合支架於體外病毒性基因療法,所分離的狗骨髓幹細胞培養將可用於未來使用同種異體骨髓幹細胞的相關狗實驗,而建立之體外慢病毒BMP2基因治療系統則提供長期給予BMP2之方法,後續將可利用本研究發展測試之組織工程組合治療系統,進一步以狗大型骨缺損模型測試其療效。
Bone grafting is often used clinically for treating osseous defects. In addition to many inherent limitations for bone grafting, its regenerative effect in large osseous defects are still less than ideal. Tissue engineering, by using scaffold, cell and growth factors, is an emergent regenerative technique with great potential. Several special concerns exist when applying tissue engineering for treating large defects. The scaffold needs sufficient mechanical strength to maintain space for regeneration. Cells with high regenerative capacity such as stem cells should be used. Gene therapy may be used for long term delivery of growth factors. The combination of proper components of tissue engineering may be needed for maximizing the regenerative effect. Previously we have developed a poly (lactide-co-glycolide) (PLGA)/collagen composite graft with good mechanical strength. Recent reports suggested that grafting with allogenic bone marrow stem cells may not induce immune and rejection response. Furthermore, the reparative and regenerative effects of the allogenic bone marrow stem cells were comparable to those of autogenic cells. Bone morphogenetic protein 2 (BMP2) is a powerful osteogenic factor. Lentiviral gene therapy has been used for long term expression of factors of interest. The purpose of this study was to establish a combined tissue engineering treatment system in preparation for the future testing in canine model with large osseous defects. The work in this study included preparation of PLGA/collagen composite scaffold, isolation canine bone marrow stem cells with stem cell phenotype, as well as construct lentiviral vector encoding BMP2 with osteoinductivity. Finally, the osteogenic effect of the established bone marrow stem cell-mediated ex vivo BMP2 lentiviral gene therapy system was proved in a SCID mice subcutaneous ectopic bone formation model. PLGA/collagen composite scaffold was used for the first time in ex vivo viral gene therapy in this study. The isolated canine bone marrow stem cells may be used in canine studies involve the using of allogenic bone marrow stem cells. The BMP2 lentiviral gene therapy system may be used for sustained delivery of BMP2 in future studies. Furthermore, the combined tissue engineering system established in this study may be used and tested in canine models with large osseous defect in the future.
目錄 i
中文摘要: v
英文摘要: vii
第一章 緒論 1
一、研究動機 1
二、組織工程 2
三、生物支架---冷凍擠壓層積成形聚乳酸-甘醇酸/膠原蛋白複合支架 2
四、冷凍擠壓層積成形 4
五、膠原蛋白 5
六、聚乳酸-甘醇酸 6
七、聚乳酸-甘醇酸/膠原蛋白複合支架 7
八、細胞---骨髓幹細胞 7
九、訊號---骨塑型蛋白 9
十、骨塑型蛋白二對骨組織的影響 10
十一、基因治療 11
十二、實驗目的 14
第二章 實驗材料與方法 15
一、實驗材料 15
二、實驗方法 25
第三章 實驗結果 44
一、構建表現骨塑型蛋白二慢病毒載體 44
二、pLKO-BMP2載體鹼性磷酸酶活性測試 45
三、狗骨髓抽取及幹細胞分離 46
四、體外測試狗骨髓幹細胞分化能力 47
五、體外pLKO-BMP2病毒感染C2C12 細胞鹼性磷酸酶活性測試 48
六、體外pLKO-BMP2病毒感染狗骨髓幹細胞之表現 49
七、掃描式電子顯微鏡觀察FCDM-PLGA/Collagen複合支架 50
八、掃描式電子顯微鏡觀察狗骨髓幹細胞貼附FCDM-PLGA與FCDM-PLGA/collagen支架之比較 51
九、X光照射分析 51
十、DEXA檢測結果 51
第四章 討論 53
結論 56
附圖 57
圖一、BMP2基因片段 57
圖二、TA-BMP2載體構建---PCR 分析 58
圖三、TA-BMP2載體---限制酶分析 59
圖四、pLKO-BMP2載體構建---PCR 分析 60
圖五、構建表現骨塑型蛋白二慢病毒載體流程 61
圖六、pLKO-BMP2載體鹼性磷酸酶活性測試 62
圖七、抽骨髓流程圖與幹細胞分離 63
圖八、油紅O染色觀察體外狗骨髓幹細胞脂肪小球形成情形 64
圖九、茜素紅染色與von Kossa染色觀察體外狗骨髓幹細胞礦化情形 65
圖十、pLKO-BMP2病毒感染C2C12細胞鹼性磷酸酶活性測試 66
圖十一、EGFP病毒感染狗骨髓幹細胞之EGFP表現 67
圖十二、pLKO-BMP2病毒感染狗骨髓幹細胞之BMP2表現 68
圖十三、pLKO-BMP2病毒感染狗骨髓幹細胞之鹼性磷酸酶活性 69
圖十四、pLKO-BMP2病毒感染狗骨髓幹細胞之骨鈣素表現 70
圖十五、FCDM-PLGA與FCDM-PLGA/collagen支架結構掃描式電子顯微鏡觀察圖 71
圖十六、狗骨髓幹細胞貼附FCDM-PLGA與FCDM-PLGA /collagen支架之掃描式電子顯微鏡觀察圖 72
圖十七、SCID小鼠皮下異位骨生成手術流程與支架外觀 73
圖十八、三週與六週X光分析 74
圖十九、三週與六週DEXA骨礦化含量(BMC)分析 75
參考文獻 76
1. Gazdag, A.R., et al., Alternatives to Autogenous Bone Graft: Efficacy and Indications. J Am Acad Orthop Surg, 1995. 3(1): p. 1-8.
2. Ehrler, D.M. and A.R. Vaccaro, The use of allograft bone in lumbar spine surgery. Clin Orthop Relat Res, 2000(371): p. 38-45.
3. Li, X.Q., et al., Differential patterns of incorporation and remodeling among various types of bone grafts. Acta Anat (Basel), 1991. 140(3): p. 236-44.
4. Brown, K.L. and R.L. Cruess, Bone and cartilage transplantation in orthopaedic surgery. A review. J Bone Joint Surg Am, 1982. 64(2): p. 270-9.
5. Petite, H., et al., Tissue-engineered bone regeneration. Nat Biotechnol, 2000. 18(9): p. 959-63.
6. Yaszemski, M.J., et al., Evolution of bone transplantation: molecular, cellular and tissue strategies to engineer human bone. Biomaterials, 1996. 17(2): p. 175-85.
7. Cowin, S.C., Tissue growth and remodeling. Annu Rev Biomed Eng, 2004. 6: p. 77-107.
8. Levenberg, S. and R. Langer, Advances in tissue engineering. Curr Top Dev Biol, 2004. 61: p. 113-34.
9. Chen, G., T. Ushida, and T. Tateishi, A biodegradable hybrid sponge nested with collagen microsponges. J Biomed Mater Res, 2000. 51(2): p. 273-9.
10. Mikos, A.G., et al., Preparation of poly(glycolic acid) bonded fiber structures for cell attachment and transplantation. J Biomed Mater Res, 1993. 27(2): p. 183-9.
11. Leong, K.F., C.M. Cheah, and C.K. Chua, Solid freeform fabrication of three-dimensional scaffolds for engineering replacement tissues and organs. Biomaterials, 2003. 24(13): p. 2363-78.
12. Sachlos, E. and J.T. Czernuszka, Making tissue engineering scaffolds work. Review: the application of solid freeform fabrication technology to the production of tissue engineering scaffolds. Eur Cell Mater, 2003. 5: p. 29-39; discussion 39-40.
13. Hutmacher, D.W., Scaffolds in tissue engineering bone and cartilage. Biomaterials, 2000. 21(24): p. 2529-43.
14. Hutmacher, D.W., J.C. Goh, and S.H. Teoh, An introduction to biodegradable materials for tissue engineering applications. Ann Acad Med Singapore, 2001. 30(2): p. 183-91.
15. Hutmacher, D.W., et al., Mechanical properties and cell cultural response of polycaprolactone scaffolds designed and fabricated via fused deposition modeling. J Biomed Mater Res, 2001. 55(2): p. 203-16.
16. Kanczler, J.M., et al., The effect of mesenchymal populations and vascular endothelial growth factor delivered from biodegradable polymer scaffolds on bone formation. Biomaterials, 2008. 29(12): p. 1892-900.
17. Yen, H.J., et al., Fabrication of precision scaffolds using liquid-frozen deposition manufacturing for cartilage tissue engineering. Tissue Eng Part A, 2009. 15(5): p. 965-75.
18. Damien, C.J., et al., Investigation of an organic delivery system for demineralized bone matrix in a delayed-healing cranial defect model. J Biomed Mater Res, 1994. 28(5): p. 553-61.
19. Chen, G., et al., The use of a novel PLGA fiber/collagen composite web as a scaffold for engineering of articular cartilage tissue with adjustable thickness. J Biomed Mater Res A, 2003. 67(4): p. 1170-80.
20. Chen, G., et al., Culturing of skin fibroblasts in a thin PLGA-collagen hybrid mesh. Biomaterials, 2005. 26(15): p. 2559-66.
21. Baksh, D., L. Song, and R.S. Tuan, Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy. J Cell Mol Med, 2004. 8(3): p. 301-16.
22. Chan, J., et al., Human fetal mesenchymal stem cells as vehicles for gene delivery. Stem Cells, 2005. 23(1): p. 93-102.
23. Caplan, A.I. and S.P. Bruder, Mesenchymal stem cells: building blocks for molecular medicine in the 21st century. Trends Mol Med, 2001. 7(6): p. 259-64.
24. Bruder, S.P. and B.S. Fox, Tissue engineering of bone. Cell based strategies. Clin Orthop Relat Res, 1999(367 Suppl): p. S68-83.
25. Castro-Malaspina, H., et al., Characterization of human bone marrow fibroblast colony-forming cells (CFU-F) and their progeny. Blood, 1980. 56(2): p. 289-301.
26. Kuznetsov, S.A., A.J. Friedenstein, and P.G. Robey, Factors required for bone marrow stromal fibroblast colony formation in vitro. Br J Haematol, 1997. 97(3): p. 561-70.
27. Piersma, A.H., et al., Migration of fibroblastoid stromal cells in murine blood. Cell Tissue Kinet, 1985. 18(6): p. 589-95.
28. Prockop, D.J., Marrow stromal cells as stem cells for nonhematopoietic tissues. Science, 1997. 276(5309): p. 71-4.
29. Conget, P.A. and J.J. Minguell, Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J Cell Physiol, 1999. 181(1): p. 67-73.
30. Caplan, E.R., et al., Diagnosis and treatment of insulin-secreting pancreatic islet cell tumors in ferrets: 57 cases (1986-1994). J Am Vet Med Assoc, 1996. 209(10): p. 1741-5.
31. Horwitz, E.M., et al., Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement. Cytotherapy, 2005. 7(5): p. 393-5.
32. Urist, M.R., Bone: formation by autoinduction. Science, 1965. 150(698): p. 893-9.
33. Wozney, J.M., et al., Novel regulators of bone formation: molecular clones and activities. Science, 1988. 242(4885): p. 1528-34.
34. Groeneveld, E.H. and E.H. Burger, Bone morphogenetic proteins in human bone regeneration. Eur J Endocrinol, 2000. 142(1): p. 9-21.
35. Ducy, P. and G. Karsenty, Genetic control of cell differentiation in the skeleton. Curr Opin Cell Biol, 1998. 10(5): p. 614-9.
36. Harland, R.M., The transforming growth factor beta family and induction of the vertebrate mesoderm: bone morphogenetic proteins are ventral inducers. Proc Natl Acad Sci U S A, 1994. 91(22): p. 10243-6.
37. Luo, J., et al., Gene therapy for bone regeneration. Curr Gene Ther, 2005. 5(2): p. 167-79.
38. Ong, J.L., et al., Osteoblast responses to BMP-2-treated titanium in vitro. Int J Oral Maxillofac Implants, 1997. 12(5): p. 649-54.
39. Bikram, M., et al., Endochondral bone formation from hydrogel carriers loaded with BMP2-transduced cells. Ann Biomed Eng, 2007. 35(5): p. 796-807.
40. Partridge, K., et al., Adenoviral BMP-2 gene transfer in mesenchymal stem cells: in vitro and in vivo bone formation on biodegradable polymer scaffolds. Biochem Biophys Res Commun, 2002. 292(1): p. 144-52.
41. Kaihara, S., et al., Simple and effective osteoinductive gene therapy by local injection of a bone morphogenetic protein-2-expressing recombinant adenoviral vector and FK506 mixture in rats. Gene Ther, 2004. 11(5): p. 439-47.
42. Park, J., et al., Bone regeneration in critical size defects by cell-mediated BMP-2 gene transfer: a comparison of adenoviral vectors and liposomes. Gene Ther, 2003. 10(13): p. 1089-98.
43. Baltzer, A.W., et al., Genetic enhancement of fracture repair: healing of an experimental segmental defect by adenoviral transfer of the BMP-2 gene. Gene Ther, 2000. 7(9): p. 734-9.
44. Park, J., et al., The effect on bone regeneration of a liposomal vector to deliver BMP-2 gene to bone grafts in peri-implant bone defects. Biomaterials, 2007. 28(17): p. 2772-82.
45. Hsu, W.K., et al., Lentiviral-mediated BMP-2 gene transfer enhances healing of segmental femoral defects in rats. Bone, 2007. 40(4): p. 931-8.
46. Miyazaki, M., et al., The effects of lentiviral gene therapy with bone morphogenetic protein-2-producing bone marrow cells on spinal fusion in rats. J Spinal Disord Tech, 2008. 21(5): p. 372-9.
47. Virk, M.S., et al., Influence of short-term adenoviral vector and prolonged lentiviral vector mediated bone morphogenetic protein-2 expression on the quality of bone repair in a rat femoral defect model. Bone, 2008. 42(5): p. 921-31.
48. Anderson, W.F., Prospects for human gene therapy. Science, 1984. 226(4673): p. 401-9.
49. Felgner, P.L. and G. Rhodes, Gene therapeutics. Nature, 1991. 349(6307): p. 351-2.
50. Levy, R.J., S.A. Goldstein, and J. Bonadio, Gene therapy for tissue repair and regeneration. Adv Drug Deliv Rev, 1998. 33(1-2): p. 53-69.
51. Kaneda, Y. and Y. Tabata, Non-viral vectors for cancer therapy. Cancer Sci, 2006. 97(5): p. 348-54.
52. Morse, M.A., Virus-based therapies for colon cancer. Expert Opin Biol Ther, 2005. 5(12): p. 1627-33.
53. Tsai, T.H., et al., Gene therapy of focal cerebral ischemia using defective recombinant adeno-associated virus vectors. Front Biosci, 2006. 11: p. 2061-70.
54. Abdellatif, A.A., et al., Gene delivery to the spinal cord: comparison between lentiviral, adenoviral, and retroviral vector delivery systems. J Neurosci Res, 2006. 84(3): p. 553-67.
55. Naldini, L., et al., In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science, 1996. 272(5259): p. 263-7.
56. Pittenger, M.F., et al., Multilineage potential of adult human mesenchymal stem cells. Science, 1999. 284(5411): p. 143-7.
57. Cheng, S.L., et al., Differentiation of human bone marrow osteogenic stromal cells in vitro: induction of the osteoblast phenotype by dexamethasone. Endocrinology, 1994. 134(1): p. 277-86.
58. Miyazaki, M., et al., The effects of lentiviral gene therapy with bone morphogenetic protein-2-producing bone marrow cells on spinal fusion in rats. J Spinal Disord Tech, 2008. 21(5): p. 372-9.
59. Sugiyama, O., et al., Lentivirus-mediated gene transfer induces long-term transgene expression of BMP-2 in vitro and new bone formation in vivo. Mol Ther, 2005. 11(3): p. 390-8.
60. Csaki, C., et al., Chondrogenesis, osteogenesis and adipogenesis of canine mesenchymal stem cells: a biochemical, morphological and ultrastructural study. Histochem Cell Biol, 2007. 128(6): p. 507-20.
61. Neupane, M., et al., Isolation and characterization of canine adipose-derived mesenchymal stem cells. Tissue Eng Part A, 2008. 14(6): p. 1007-15
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關論文
 
無相關期刊
 
1. 探討c-Jun 胺基末端激酶(JNK)在骨型態發生蛋白(BMP2)誘發之成骨細胞分化過程中所扮演的角色
2. 以局部基因治療方法合併給予骨形成蛋白四號及血管內皮生長因子對顱顏骨缺損的影響
3. 分析WNT-1, BMP2及EGFR啟動子之G-quadruplex形成序列
4. 超音波刺激三維空間培養之間葉幹細胞誘發BMP2及sRANKL增益成骨細胞之生成
5. 合併應用同種異體骨髓基質細胞為媒介之慢病毒骨形成蛋白二基因治療及聚乳酸-甘醇酸/膠原蛋白支架於垂直骨擴增術之狗研究
6. 成骨細胞分化調控之分子機轉研究
7. 幾丁聚醣/褐藻醣膠奈米粒控制釋放鹼性纖維母細胞生長因子在神經組織工程之應用
8. 骨內骨形態發生蛋白四腺病毒基因治療對牙科植體早期穩定度之影響
9. HIF-1α促進造骨細胞內BMP-2表現之機轉
10. 骨型態發生蛋白的訊息途徑對細胞功能之探討:BMP-4和流體剪力對骨母細胞分化作用及腫瘤細胞生長作用之影響
11. 含奈米級氫氧基磷灰石與聚己內酯之電紡奈米纖維膜製備與在骨組織工程之應用
12. 骨髓幹細胞及冷凍擠壓層積成型聚乳酸-甘醇酸強化骨形態發生蛋白4/血管內皮生長因子165膠原蛋白活化基因基質之協同骨生成作用
13. 1. 以二甲基甲醯胺調控醣基化反應合成2,6-雙去氧-a-醣苷鍵,並延伸至一鍋化、迭代醣基化反應。2. 利用保護基Picolinoyl -對醣受體產生遠端引導效應合成去氧-b-醣苷鍵
14. 利用PCS生物反應器以Rhizopus oligosporus NTU 5進行黑豆漿發酵並探討異黃酮去醣基化效率及生理活性之研究
15. An Understanding of the O2-Iron Protoporphyrin IX Binding in Human Serum Albumin and its Engineered Mutant from the O2 Diffusion Pathways and Escape Routes