細胞治療在組織工程領域上，為一相當具有前瞻性的治療方法。曾有研究群利用注射幹細胞進行組織再生的治療，但其效果有限。原因在於收集細胞時用酵素脫附細胞的動作，會破壞細胞與細胞間的連結性，因此可能會影響後續細胞移植的治療效果。本實驗室先前已開發出細胞球體生產系統，利用細胞不會貼附在甲基纖維素水膠的特性，製備大小均一且可經由針頭注射的細胞球體。實驗結果顯示，細胞球體具有完整的細胞外間質及黏附性蛋白，注射後，嵌入肌肉間隙中並黏附於新生環境裡，而有效的留置於體內。本研究利用人類臍帶血間葉幹細胞(human cord-blood mesenchymal stem cells, cbMSC)與人類臍帶靜脈內皮細胞(human umbilical vein endothelial cells, HUVEC)在體外共同培養成3-D核層細胞球體，並探討此一核層細胞球體的增生、分佈及最佳化等特性。體外實驗部分，我們先行探討cbMSC/HUVEC核層細胞球體的形成機制，結果發現當cbMSC作為核時，才可形成核層細胞球體。另外我們藉由類血管網路形成(Tube Formation Assay)評估cbMSC/HUVEC核層細胞球體分化血管之能力及探討 cbMSC/HUVEC 細胞數比例是否會影響類血管網路形成能力。結果顯示以 cbMSC 與 HUVEC 比例1：1其類血管網路結構形成的速度及完整性是最好的。我們也分析了SMA、SM22、MYH10、VEGF、TGF-β1、Ang1-1此六種與血管形成相關的基因，除TGF-β1外，其餘五種基因的表現量隨時間增長有上升的趨勢。體內實驗部分，我們把cbMSC/HUVEC核層細胞球體與Matrigel混合，進行老鼠背部皮下注射，探討其分部、增生及是否有形成血管網路之情形。結果顯示，cbMSC/HUVEC核層細胞球體於7天後已部份分化為血管，並與宿主血管連結，證明其確實有分化為血管之能力。

1. Gulbins, H.;Meiser, B. M.;Reichenspurner, H., et al. "Cell transplantation - A potential therapy for cardiac repair in the future?" Heart Surgery Forum 5, E28-E34 (2002).
2. Anversa, P., Nadal-Ginard, B., “Myocyte renewal and ventricular remodelling,” Nature, 415, pp.240-243, 2002.
3. Tomita, S., Mickle, D.A., Weisel, R.D., Jia, Z.Q., Tumiati, L.C., Allidina, Y., Liu, P., Li, R.K., “Improved heart functions with myogenesis and angiogenesis after autologous porcine bone marrow stromal cell transplantation,” J Thorac Cardiovasc Surg, 123, pp.1132-1140, 2002.
4. Davani, S., Marandin, A., Mersin, N., Royer, B., Kantelip, B., Hervé, P., Etievent, J.P., Kantelip, J.P., “Mensenchymal progenitor cells differentiate into an endothelial phenotype, enhance vascular density, and improve heart function in rat cellular cardiomyoplasty model,” Circulation, 108, suppl. 1, pp.II253-258, 2003.
5. Saito, T., Kuang, J.Q., Lin, C.C., Chiu, R.C., “Transcoronary implantation of bone marrow stromal cells ameliorates cardiac function after myocardial infarction,” J Thorac Cardiovasc Surg, 126, pp.114-123, 2003.
6. Jackson, K.A., Majka, S.M., Wang, H., Pocius, J., Hartley, C.J., Majesky, M.W., Entman, M.L., Michael, L.H., Hirschi, K.K., Goodell, M.A. “Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells,” J Clin Invest, 107, pp.1395-1402, 2001.
7. Reffelmann, T., Kloner, R.A., “Cellular cardiomyoplasty--cardiomyocytes, skeletal myoblasts, or stem cells for regenerating myocardium and treatment of heart failure?”Cardiovasc Res, 58, pp.358-368, 2003.
8. Laflamme, M.A., Murry, C., “Regenerating the heart,” Nat Biotechnol, 23, pp.845-856, 2005.
9. Hsih, P.C., Davis, M.E., Lisowski, L.K., Lee, R.T., “Endothelial-cardiomyocyte interactions in cardiac development,” Annu Rev Physiol, 68, pp.51-66, 2006.
10. Maintenance of the culture cell lines (1994) in Culture of animal cells, eds Freshney RI (Wiley-Liss, New York), pp153-154.
11. Shimizu, T., Yamato, M., Kikuchi, A., Okano, T., “Cell sheet engineering for myocardial tissue reconstruction,” Biomaterials, 24, pp.2309-2316, 2003.
12. Rosenstrauch, D., Poglajen, G., Zidar, N., Gregoric, I.D., “Stem celltherapy for ischemic heart failure,” Tex Heart Inst J, 32, pp.339–347, 2005.
13. Fukuda, K. “Progress in myocardial regeneration and cell transplantation” Circ J, 69, pp.1431-1446, 2005.
14. Teng, C.J., Luo, J., Chiu, R.C., Shum-Tim D., “Massive mechanical loss of microspheres with direct intramyocardial injection in the beating heart: implications for cellular cardiomyoplasty,” J Thorac Cardiovasc Surg, 132, pp.628-632, 2006.
15. Hsieh, P.C., Davis, M.E., Gannon, J., MacGillivray, C., Lee R.T., “Controlled delivery of PDGF-BB for myocardial protection using injectable self-assembling peptide nanofibers,” J Clin Invest, 116, pp.237-248, 2006.
16. Grafflin MW, “Derivatives of Cellulose,” Cellulose and Cellulose Derivatives, (1963) 930-937.
17. 賀孝雍, “有機化學”, 曉園出版公司, (1989) 台北, 394.
18. Qiu, Y.;Park, K. "Environment-sensitive hydrogels for drug delivery." Advanced Drug Delivery Reviews 53, 321-339 (2001).
19. Haque, A.;Richardson, R. K.;Morris, E. R., et al. "Thermogelation of methylcellulose: 2 effect of hydroxypropyl substituents." Carbohydrate Polymers 22, 175-186 (1993).
20. Jeong, B.;Kim, S. W.;Bae, Y. H. "Thermosensitive sol-gel reversible hydrogels." Advanced Drug Delivery Reviews 54, 37-51 (2002).
21. Yang, M.J., Chen, C.H., Lin, P.J., Huang. C.H., Chen, W., Sung, H.W., “Novel method of forming human embryoid bodies in a polystyrene dish surface-coated with a temperature-responsive methylcellulose hydrogel,” Biomacromolecules, 8, pp.2746-2752, 2007.
22. Lee, W.Y., Chang, Y.H., Yeh, Y.C., Chen, C.H., Lin, K.M., Huang, C.C., Chang, Y., Sung, H.W., “The use of injectable spherically-symmetric cell aggregates self-assembled in a thermo-responsive hydrogel system for enhanced cell transplantation, ” Biomaterials, 30, pp.5505-5513, 2009.
23. Lee, S.H., Wolf PL , Escudero R, “Early expression of angiogenesis factors in acute myocardial ischemia and infarction”N Engl J Med ,pp3429 -626,2000.
24. Krenning G, van Luyn MJ, Harmsen MC “Endothelial progenitor cell-based neovascularization: implications for therapy” Trends Mol Med. ,pp.180-9, 2009.
25. Staton, C.A., Stribbling, S.M., Tazzyman, S., Hughes, R., Brown, N.J., Lewis, C.E., “Current methods for assaying angiogenesis in vitro and in vivo,” Int J Exp Patho, 85, pp.233-248, 2004.
26. Kubota, Y., Kleinman, H.K., Martin, G.R., Lawley, T.J., “Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures,” J Cell Biol, 107, pp.1589-1598, 1998.
27. Koike, N., Fukumura, D., Gralla, O., Au, P., Schechner, J.S., Jain, R.K., “Tissue engineering: creation of long-lasting blood vessels,” Nature, 428, pp.138-139, 2004.
28. Nör, J.E., Peters, M.C., Christensen, J.B., Sutorik, M.M., Linn, S., Khan, M.K., Addison, C.L., Mooney, D.J., Polverini, P.J., “Engineering and characterization of functional human microvessels in immunodeficient mice,” Lab Invest, 81, pp.453-463, 2001
29. Schechner, J.S., Nath, A.K., Zheng, L., Kluger, M.S., Hughes, C.C., Sierra-Honigmann, M.R., Lorber, M.I., Tellides, G., Kashgarian, M., Bothwell, A.L., Pober, J.S., “In vivo formation of complex microvessels lined by human endothelial cells in an immunodeficient mouse,” Proc Natl Acad Sci U S A, 97, pp.9191-9196, 2000.
30. Au, P., Tam, J., Fukumura, D., Jain, R.K., “Bone marrow-derived mesenchymal stem cells facilitate engineering of long-lasting functional vasculature,” Blood, 111, pp.4551-4558, 2008.
31. Jain, R.K., “Molecular regulation of vessel maturation,” Nat Med, 9, pp.685-693, 2003.
32. Hirschi, K.K., Rohovsky, S.A., D'Amore, P.A., “PDGF, TGF-beta, and heterotypic cell-cell interactions mediate endothelial cell-induced recruitment of 10T1/2 cells and their differentiation to a smooth muscle fate,” J Cell Biol, 141, pp.805-814, 1998.
33. Melero-Martin, J.M., De Obaldia, M.E., Kang, S.Y., Khan, Z.A., Yuan, L., Oettgen, P., Bischoff, J., “Engineering robust and functional vascular networks in vivo with human adult and cord blood-derived progenitor cells,” Circ Res, 103, pp.194-202, 2008.