過去在血管新生的研究中，多為利用組織切片得到平面相關資訊，如此僅能觀察到局部的現象。為了得到更清楚的立體空間結構證據，因此在本實驗中，我們使用了共軛焦雷射掃瞄式顯微鏡術重建新生血管的內部結構，做進一步的觀察。本研究主要的目的是，觀察bFGF與從中藥〝人蔘〞裡萃取純化出來的非蛋白質類新型血管增生因子ginsenoside Rg1 (Rg1)，在體內促進血管新生的情形。實驗裡以能表現綠色螢光的基因轉殖鼠做為實驗動物，並利用共軛焦雷射掃瞄式顯微鏡技術以3D影像重建血管新生情況，觀察血管新生情形。
本研究主要分為體外實驗以及體內實驗兩部分進行探討。在體外實驗部分，我們以細胞培養測試的方式，評估在37℃下保存不同天數的bFGF以及Rg1對人類臍帶靜脈內皮細胞(HUVECs)增殖能力的影響。當bFGF於37℃下保存的天數越長，對於HUVECs增殖能力下降越明顯；而Rg1在37℃下保存30天的過程中，其對於HUVECs增殖能力沒有明顯的差異。
第二部分的體內實驗裡，我們將bFGF與Rg1和growth factor reduced matrigel均勻摻混，接著注射入老鼠腹部皮下形成plug，於不同時間點取樣觀察bFGF與Rg1對於誘導生物體內血管新生的影響。實驗裡，分別在注射後第1、4、6、8個星期進行取樣，分析方法分為三部份：共軛焦雷射掃瞄式顯微鏡術分析、病理組織切片分析、血紅素含量分析等。以共軛焦雷射掃瞄式顯微鏡術觀察matrigel plug內的血管新生情形，將新生血管的螢光以立體空間方式完整重建；利用病理組織切片評估免疫反應，並且以Image Pro® system計算matrigel plug內血管新生的密度、滲入深度及脂肪細胞滲入深度。
我們不僅由常用的組織切片獲得平面相關資訊，而且能夠利用共軛焦雷射掃瞄式顯微鏡術做為觀察matrigel中新生血管生長情況，將新生血管以螢光方式重建成具有立體空間結構的影像，獲得更具體的血管新生空間分布的證據。
本研究同時也觀察到，無論是bFGF或者是Rg1，用以體內促進血管新生時，只要當生物組織再生達到一定程度時，初期經由血管增生因子所誘導的多數新生血管開始進行血管萎縮的調控，將新生血管數目調整為再生組織所需要的新生血管數量。
上述的實驗結果顯示，我們成功建立以共軛焦雷射掃描式顯微鏡術觀察血管新生的技術，突破傳統組織切片獲得的平面相關資訊，得到新生血管在植入材料中的分布情形，獲得更具體的證據。

1. Scandurra R, Consalvi V, Chiaraluce R, Politi L, Engel PC. Protein stability in extremophilic archaea. Frontiers in Bioscience 2000;5:787-795.
2. Passaniti A, Taylor RM, Pili R, Guo Y, Long PV, Haney JA, Pauly RR, Grant DS, Martin GR. A simple, quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement membrane, heparin, and fibroblast growth factor. Lab Invest 1992;67:519-528.
3. Vacanti JP, Vacanti CA. The challenge of tissue engineering. In: Lanza RP, Langer R, Chick WL, editors. Principles of tissue engineering. R.G. Landes Company, Austin: Academic Press, 1997. p. 1-5.
4. Langer R. Tissue engineering. Science 1993□260:920-926.
5. Tabata Y. Necessity of drug delivery dystems to tissue engineering. In: Park KD, Kwon IC, Yui N, Jeong SY, Park K, editors. Biomaterials and drug delivery toward new mellenium. Korea: Han Rim Won Publishing Co., 2000. p. 531-544.
6. Kofidis T, Akhyari P, Boublik J, Theodorou P, Martin, U, Ruhparwar A, Fischer S, Eschenhagen T, Kubis HP, Kraft T, Leyh R, Haverich A. In vitro engineering of heart muscle: artificial myocardial tissue. J Thorac Cardiov Sur 2002□124:63-69.
7. Li RK, Yau TM, Weisel RD, Mickle DA, Sakai T, Choi A, Jia ZQ. Construction of a bioengineered cardiac graft. J Thorac Cardiov Sur 2000□119:368-375.
8. Freed LE, Vunjak-Novakovic G. Tissue culture bioreactors: chondrogenesis as a model system. In: Lanza RP, Langer R, Chick WL, editors. Principles of tissue engineering. R.G. Landes Company, Austin: Academic Press, 1997. p.151-65.
9. Chapekar MS. Tissue engineering: challenges and opportunities. J Biomed Mater Res 2000□53:617-620.
10. Soker S, Machado M. Atala A. Systems for therapeutic angiogenesis in tissue engineering. World J Urol 2000□18:10-18.
11. Heldin CH, Usuki K, Miyazono K. Platelet-derived endothelial cell growth factor. J Cell Biochem 1991□47:208-210.
12. Kawai K, Suzuki S, Tabata Y, Ikada Y, Nishimura Y. Accelerated tissue regeneration through incorporation of basic fibroblast growth factor-impregnated gelatin microspheres into artificial dermis. Biomaterials 2000□21:489-499.
13. Morisaki N, Watanabe S, Tezuka M, Zenibayashi M, Shiina R, Koyama N, Kanzaki T, Saito Y. Mechanism of angiogenic effects of saponin from ginseng Radix rubra in human umbilical vein endothelial cells. Brit J Pharmacol 1995□115:1188-1193.
14. Kalluri R. Basement membranes: Structure, assembly and role in tumour angiogenesis. Nat Rev Cancer 2003;3:422-433.
15. Gospodarowicz D, Massoglia S, Cheng J, Fujii DK. Effect of fibroblast growth factor and lipoproteins on the proliferation of endothelial cells derived from bovine adrenal cortex, brain cortex and corpus luteum capillaries. J Cell Physiol 1986;127:121-136.
16. Esch F, Baird A, Ling N, Ueno N, Hill F, Deneroy L, Klepper R, Gospodarowicz D, Bohlen P, Guillemin R. Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino terminal sequence of bovine brain acidic FGF. Proc Natl Acad Sci USA 1985;85:6507-6511.
17. Folkman J, Klagsburn M. Angiogenic factors. Science 1987;235:442-447.
18. Tsuboi R, Rifkin DB. Recombinant basic fibroblast growth factor stimulates wound healing in healing-impaired db/db mice. J Exp Med 1990;172:245-251.
19. Vemuri S, Beylin I, Sluzky V, Stratton P, Eberlein G, Wang YJ. The stability of bFGF against thermal denaturation. J Pharm Pharmacol 1994;46:481-486.
20. Gospodarowicz D, Cheng J. Heparin protects basic and acidic FGF from inactivation. J Cell Physiol 1986;128:475-484.
21. Foster LC, Thompson SA, Tarnowski SJ. Methods and formulations for stabilizing fibroblast growth factor. International Patent Number WO91/15509,1991.
22. Liao B, Newmark H, Zhou R. Neuroprotective effects of ginseng total saponin and ginsenosides Rb1 and Rg1 on spinal cord neurons in vitro. Exp Neuro 2002□173:224-234.
23. Scott GI, Colligan PB, Ren BH, Ren J. Ginsenosides Rb1 and Re decrease cardiac contraction in adult rat ventricular myocytes: role of nitric oxide. Brit J Pharmacol 2001□134:1159-1165.
24. 恩斯特˙D˙普林曾柏格. 人參：永保青春和活力. 台北縣: 小薰書房, 1999.
25. 張維懋, 王致權. 21世紀癌症新剋星：人參皂苷. 台北市: 愛克斯文化, 2002.
26. Liu M, Zhang JT. Effects of ginsenoside Rg1 on c-fos gene expression and cAMP levels in rat hippocampus. Pharmacologica Sinica 1996□17:171-174.
27. Tong LS, Chao CY. Effects of ginsenoside Rg1 of Panax ginseng on mitosis in human blood lymphocytes in vitro. Am J Chinese Med 1980□8:254-267.
28. Nah SY, Oh S. Modulation of G protein alpha-subunit mRNA levels in discrete rat brain regions by cerebroventricular infusion of ginsenoside Rc and Rg1. Neurochem Res 2003□28:691-697.
29. Lee YJ, Chung E, Lee KY, Lee YH, Huh B, Lee SK. Ginsenoside-Rg1, one of the major active molecules from Panax ginseng, is a functional ligand of glucocorticoid receptor. Mol Cell Endocrinol 1997□133:135-140.
30. Losordo DW, Isner JM. Estrogen and angiogenesis: a review. Arterioscl Throm Vas 2001□21:6-12.
31. Cid MC, Schnaper HW, Kleinman HK. Estrogens and the vascular endothelium. Annals NY Acad Sci 2002□966:143-157.
32. Chan RY, Chen WF, Dong A, Guo D, Wong MS. Estrogen-like activity of ginsenoside Rg1 derived from Panax notoginseng. J Clin Endocr Metab 2002□87:3691-3695.
33. Morbidelli L, Donnini S, Ziche M. Role of nitric oxide in the modulation of angiogenesis. Curr Pharm Design 2003□9:521-530.
34. Babaei S, Stewart DJ. Overexpression of endothelial NO synthase induces angiogenesis in a co-culture model. Cardiovasc Res 2002□55:190-200.
35. Miyamoto E, Odashima S, Kitagawa I, Tsuji A. Stability kinetics of ginsenosides in aqueous solution. J Pharm Sci 1984□73:409-410.
36. Kitagawa I, Taniyama T, Yoshikawa M, Ikenishi Y, Nakagawa Y. Chemical studies on crude drug processing. VI. Chemical structures of malonyl-ginsenosides Rb1, Rb2, Rc, and Rd isolated from the root of Panax ginseng C. A. Meyer. Chem Pharm Bull 1989□37:2961-2970.
37. 陳榮福.中藥藥理學. 台北縣: 國立中國醫藥研究所, 1991. p. 113-264.
38. Huang YC, Chen CT, Chen SC, Lai PH, Liang HC, Chang Y, Yu LC, Sung HW. A natural compound (ginsenoside Re) isolated from Panax Ginseng as a novel angiogenic agent for tissue regeneration. Pharm Res 2005;22:636-646.
39. Liang HC, Chen CT, Chang Y, Huang YC, Chen SC, Sung HW. Loading of a novel angiogenic agent, ginsenoside Rg(1) in an acellular biological tissue for tissue regeneration. Tissue Eng 2005;11:835-846.
40. Yu LC, Chen SC, Chang WC, Huang YC, Lin KM, Lai PH, Hsiao W WW, Sung HW. Stability and activity of novel angiogenic agents, ginsenoside Rg1 and Re, isolated from Panex ginseng. Int J Pharm submitted.
41. Sengupta S, Toh SA, Sellers LA, Skepper JN, Koolwijk P, Leung HW, Yeung HW, Wong RN, Sasisekharan R, Fan TP. Modulating angiogenesis: the yin and the yang in ginseng. Circulation 2004;110:1219-1225.
42. Nana R. Taking the confusion out of confocal microscopy. BioTeach Journal 2003;1:75-80.
43. Qiu LR, Ding XM, Liu J. Confocal Measurement Approach for enhancing lateral resolution using a phase-only pupil. J Phys: Conf Ser 2005;13:422-425.
44. Hasim A, Erin BD, Robert A. The sponge/Matrigel angiogenesis assay. Angiogenesis 2002;5:75-80.
45. Hagan S, Hiscott P, Sheridan CM, Wong D, Grierson I, McGalliard J. Effects of the matricellular protein SPARC on human retinal pigment epithelial cell behavior. Mol Vis 2003;9:87-92.
46. Wajih N, Sane DC. Angiostatin selectively inhibits signaling by hepatocyte growth factor in endothelial and smooth muscle cells. Blood 2003□101:1857-1863.
47. Riss TL, Moravec RA. Comparison of MTT, XTT, and a novel tetrazolium compound MTS for in vitro proliferation and chemosensitivity assays. Mol Biol Cell 1992;3(suppl):184.
48. Berridge MV, Tan AS. Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction. Arch Biochem Biophys 1993;303:474-482.
49. Carolyn AS, Stephen MS, Simon T, Russell H, Nicola B, Claire EL. Current methods for assaying angiogenesis in vitro and in vivo. Int J Exp Path 2004;85:233-248.
50. Hideyoshi T, Atsunori N, Donna BS, Anna JR, Michael AN, Joao SN, Takashi K, Anthony JD, Noriko M. 3D-confocal structural analysis of bone marrow-derived renal tubular cells during renal ischemia/reperfusion injury. Lab Invest 2006;86:72-82.
51. Marie P, James H, Yiwen L, Angel S, William O, Karen K, Jay O, Andrea H, Bronislaw P, Larry W, Peter B, Daniel JH. Antivascular endothelial growth factor receptor (fetal liver kinase 1) monoclonal antibody inhibits tumor angiogenesis and growth of several mouse and human tumor. Cancer Res 1999;59:5209-5218.
52. Alberto D. Confocal and two-photon microscopy: Foundations, Applications, and Advances. Wiley-Liss, Inc., New York. 2002. p. 115-116.
53. Crosby WH, Munn JI, Furth FW. Standardizing a method for clinical hemoglobinometry. US Armed Forces Med J 1954;5:693.
54. Nobuko K, Kazuhiro T, Eleni NL, Kazuhiko I, Shuhei T, Yasuo K. De novo adipogenesis in mice at the site of injection of basement membrane and basic fibroblast growth factor. Proc Natl Acad Sci USA 1998;95:1062-1066.
55. Yu K, Makoto O, Takashi I, Yasuhiko T. Adipose tissue engineering based on human preadipocytes combined with gelatin microspheres containing basic fibroblast growth factor. Biomaterials 2003;24:2513-2521.
56. Peter C. Angiogenesis in health and disease. Nat Med 2003;9:653-660.
57. Rakesh KJ. Molecular regulation of vessel maturation. Nat Med 2003;9:685-693.
58. Beat AI, Michel AL. Angiogenesis and inflammation face off. Nat Med 2006;12:171-172.