臺灣博碩士論文加值系統

在血管新生發生的過程中，基質金屬蛋白��2號(MMP-2)常被促進以分解基底膜，然後細胞會延著基底膜打開的洞，朝向血管刺激原移動。腫瘤要開始長出新的血管前，基質金屬蛋白��2號是重要的「angiogenic switch」，它對內皮細胞的移動有重要的調控作用。所以我想要了解，中草藥對於內皮細胞移動的調控是否透過基質金屬蛋白��2號來執行。基質金屬蛋白��2號主要是分解type IV collagen，又叫gelatinase A，其酵素活性分析方法，將基質金屬蛋白��2號的substrate gelatin加到SDS- polyacrylamide gel內，細胞分泌液經電泳分離後，含有基質金屬蛋白��2號的位置將分解gelatin，染coomassie blue後，gelatin被分解的區域會呈現空白狀態。讀取空白區域的密度後，進行統計分析。Calf pulmonary artery endothelial cells (CPAE)與中草藥作用6小時後，收集其狀況液(conditioned medium)分析其酵素活性。使用的中草藥有40種，其中會促進內皮細胞移動的有9種，對內皮細胞移動沒有影響的有9種，會抑制內皮細胞移動的有22種。促進細胞移動的中草藥中有4個會促進基質金屬蛋白��2號的活性，有1個會抑制基質金屬蛋白��2號的活性，有4個不影響基質金屬蛋白��2號的活性；不影響細胞移動的中草藥中有1個會促進基質金屬蛋白��2號的活性，有2個會抑制基質金屬蛋白��2號的活性，有6個不影響基質金屬蛋白��2號的活性；會抑制細胞移動的中草藥中有1個會促進基質金屬蛋白��2號的活性，有11個會抑制基質金屬蛋白��2號的活性，有10個不影響基質金屬蛋白��2號的活性，這些結果顯示，可能因為中草藥成分的組成是多重的，所以很難只連結單一的功能。

In the process of angiogenesis, the MMP-2 plays an important role in basement membrane degradation to facilitate cell migration. Especially in tumorgenesis, it has been shown that MMP-2 is an important “angiogenic switch”. It has important regulation in endothelial cell migration. Now I am interested in evaluating the hypothesis that Chinese herbs that affect endothelial cells migration may be mediated though MMP-2. The MMP-2 activity is assayed by SDS-polyacrylamide gel with gelatinas substrate. The conditioned medium collected from cells treated with Chinese herbs was separated by electrophoresis. After staining with Coomassie Blue, The degradation of gelatin, as indicated by the blank gel, was estimated by reading the density of the blank area. CPAE(calf pulmonary artery endothelial cells ) in PBS were treated with herbs in various concentrations for 6 hr. Our results showed that 40 herbs had various potencies to inhibit MMP-2 activity: in the group that enhances migration, 4 herbs enhanced, one inhibited, and 4 with no effect; in the group that with no effect, 1enhanced, 2 inhibited, and 6 with no effect; and in the group that inhibits migration, 1 enhanced, 11 inhibited, and 10 with no effect. These results suggest that there is no one simple functional correlation with Chinese herbs can be identified, probably due to its multiconsituents nature.

中文摘要 1
英文摘要 3
目錄 4
表目錄 7
圖目錄 8
縮寫檢索表 9
緒論 11
　1 中草藥的應用 11
　2 中草藥和血管新生 11
　3 血管新生的形成及其作用 12
　4 所使用的中草藥分類及過去的研究 13
　5 matrix metalloproteinase 2 (MMP-2) 16
　6 影響內皮細胞migration的可能原因 17
　7 研究假設 17
材料與方法 20
實驗材料 20
A 試劑藥品 20
B 儀器 21
實驗方法 22
1 上皮細胞株繼代培養 22
　1-1 細胞培養 22
　1-2 細胞保存 23
　1-3 細胞解凍 24
　1-4 細胞計數 24
2 基質金屬蛋白�﹞妞〝吨尷R 25
　2-1 sample製備 25
　2-2 Gelatin SDS-PAGE製作 26
　2-3 Gelatin SDS-PAGE膠體電泳 27
　2-4 Coomassie Blue染色 28
3 細胞凋亡分析 29
　3-1 sample製備 29
　3-2 細胞凋亡分析 29
3-3 細胞存活分析 30
4統計與密度分析 30
　4-1密度分析法 30
　4-2統計分析法 30
結果與討論 31
1、會促進CPAE migration的中草藥，對MMP-2活性的影響。 31
　2、不影響CPAE migration的中草藥，對MMP-2活性的影響。 32
　3、抑制CPAE migration的中草藥，對MMP-2活性的影響。 34
　4、會影響MMP-2活性的中草藥，可能藉由造成細胞的死亡來影響MMP-2的活性。 36
　5、中草藥影響cell migration和MMP-2活性關係 36
參考文獻 38
表 41
圖 45

1.http://baike.baidu.com/view/14724.htm.
2.http://baike.baidu.com/view/113557.htm.
3.http://zh.wikipedia.org/wiki/中草葯
4.http://en.wikipedia.org/wiki/Ephedra.
5.http://en.wikipedia.org/wiki/Artemisinin.
6.Fan, T.P., et al., Angiogenesis: from plants to blood vessels. Trends Pharmacol Sci, 2006. 27(6): p. 297-309.
7.Wani, M.C., et al., Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. J Am Chem Soc, 1971. 93(9): p. 2325-7.
8.Avramis, I.A., R. Kwock, and V.I. Avramis, Taxotere and vincristine inhibit the secretion of the angiogenesis inducing vascular endothelial growth factor (VEGF) by wild-type and drug-resistant human leukemia T-cell lines. Anticancer Res, 2001. 21(4A): p. 2281-6.
9.Escuin, D., E.R. Kline, and P. Giannakakou, Both microtubule-stabilizing and microtubule-destabilizing drugs inhibit hypoxia-inducible factor-1alpha accumulation and activity by disrupting microtubule function. Cancer Res, 2005. 65(19): p. 9021-8.
10.Liekens, S., E. De Clercq, and J. Neyts, Angiogenesis: regulators and clinical applications. Biochem Pharmacol, 2001. 61(3): p. 253-70.
11.Lay, I.S., et al., Crude extract of Salvia miltiorrhiza and salvianolic acid B enhance in vitro angiogenesis in murine SVR endothelial cell line. Planta Med, 2003. 69(1): p. 26-32.
12.Kok, T.W., et al., The anti-angiogenic effect of sinomenine. Angiogenesis, 2005. 8(1): p. 3-12.
13.Zhou, H.J., et al., Artesunate inhibits angiogenesis and downregulates vascular endothelial growth factor expression in chronic myeloid leukemia K562 cells. Vascul Pharmacol, 2007. 47(2-3): p. 131-8.
14.Shyu, K.G., et al., Saikosaponin C induces endothelial cells growth, migration and capillary tube formation. Life Sci, 2004. 76(7): p. 813-26.
15.Folkman, J., What is the role of endothelial cells in angiogenesis? Lab Invest, 1984. 51(6): p. 601-4.
16.Folkman, J. and Y. Shing, Angiogenesis. J Biol Chem, 1992. 267(16): p. 10931-4.
17.Gross, J.L., D. Moscatelli, and D.B. Rifkin, Increased capillary endothelial cell protease activity in response to angiogenic stimuli in vitro. Proc Natl Acad Sci U S A, 1983. 80(9): p. 2623-7.
18.Ausprunk, D.H. and J. Folkman, Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis. Microvasc Res, 1977. 14(1): p. 53-65.
19.Folkman, J. and C. Haudenschild, Angiogenesis in vitro. Nature, 1980. 288(5791): p. 551-6.
20.Nguyen, M., J. Folkman, and J. Bischoff, 1-Deoxymannojirimycin inhibits capillary tube formation in vitro. Analysis of N-linked oligosaccharides in bovine capillary endothelial cells. J Biol Chem, 1992. 267(36): p. 26157-65.
21.Benjamin, L.E., I. Hemo, and E. Keshet, A plasticity window for blood vessel remodelling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF. Development, 1998. 125(9): p. 1591-8.
22.Handsley, M.M., THE MATRIX METALLOPROTEINASES AND THEIR INHIBITORS.
23.Nguyen, M., J. Arkell, and C.J. Jackson, Human endothelial gelatinases and angiogenesis. Int J Biochem Cell Biol, 2001. 33(10): p. 960-70.
24.Bischoff, J., Cell adhesion and angiogenesis. J Clin Invest, 1997. 100(11 Suppl): p. S37-9.
25.Rundhaug, J.E., Matrix metalloproteinases and angiogenesis. J Cell Mol Med, 2005. 9(2): p. 267-85.
26.Zachary, I. and G. Gliki, Signaling transduction mechanisms mediating biological actions of the vascular endothelial growth factor family. Cardiovasc Res, 2001. 49(3): p. 568-81.
27.Kobayashi, M., et al., MAPKAPK-2-mediated LIM-kinase activation is critical for VEGF-induced actin remodeling and cell migration. Embo J, 2006. 25(4): p. 713-26.
28.Abedi, H. and I. Zachary, Signalling mechanisms in the regulation of vascular cell migration. Cardiovasc Res, 1995. 30(4): p. 544-56.
29.Gurjar, M.V., R.V. Sharma, and R.C. Bhalla, eNOS gene transfer inhibits smooth muscle cell migration and MMP-2 and MMP-9 activity. Arterioscler Thromb Vasc Biol, 1999. 19(12): p. 2871-7.
30.Deryugina, E.I., et al., Matrix metalloproteinase-2 activation modulates glioma cell migration. J Cell Sci, 1997. 110 ( Pt 19): p. 2473-82.
31.McClung, H.M., et al., SPARC upregulates MT1-MMP expression, MMP-2 activation, and the secretion and cleavage of galectin-3 in U87MG glioma cells. Neurosci Lett, 2007. 419(2): p. 172-7.
32.Zhuge, Y. and J. Xu, Rac1 mediates type I collagen-dependent MMP-2 activation. role in cell invasion across collagen barrier. J Biol Chem, 2001. 276(19): p. 16248-56.
33.Giannelli, G., et al., Induction of cell migration by matrix metalloprotease-2 cleavage of laminin-5. Science, 1997. 277(5323): p. 225-8.
34.Hotary, K., et al., Regulation of cell invasion and morphogenesis in a three-dimensional type I collagen matrix by membrane-type matrix metalloproteinases 1, 2, and 3. J Cell Biol, 2000. 149(6): p. 1309-23.
35.Peng, B., et al., Suppression of human ovarian SKOV-3 cancer cell growth by Duchesnea phenolic fraction is associated with cell cycle arrest and apoptosis. Gynecol Oncol, 2008. 108(1): p. 173-81.
36.Park, W.H., et al., Tumor initiation inhibition through inhibition COX-1 activity of a traditional Korean herbal prescription, Geiji-Bokryung-Hwan, in human hepatocarcinoma cells. Immunopharmacol Immunotoxicol, 2005. 27(3): p. 473-83.
37.Li, R.W., et al., Anti-inflammatory activity of Chinese medicinal vine plants. J Ethnopharmacol, 2003. 85(1): p. 61-7.
38.Li, H., et al., Triptolide inhibits proinflammatory factor-induced over-expression of class II MHC and B7 molecules in renal tubular epithelial cells. Acta Pharmacol Sin, 2002. 23(9): p. 775-81.
39.Hu, K.B., et al., Inhibition of vascular endothelial growth factor expression and production by triptolide. Planta Med, 2002. 68(4): p. 368-9.
40.Wang, B., et al., Triptolide, an active component of the Chinese herbal remedy Tripterygium wilfordii Hook F, inhibits production of nitric oxide by decreasing inducible nitric oxide synthase gene transcription. Arthritis Rheum, 2004. 50(9): p. 2995-303.
41.Sigstedt, S.C., et al., Evaluation of aqueous extracts of Taraxacum officinale on growth and invasion of breast and prostate cancer cells. Int J Oncol, 2008. 32(5): p. 1085-90.
42.Kim, K.W., et al., Polygonum cuspidatum, compared with baicalin and berberine, inhibits inducible nitric oxide synthase and cyclooxygenase-2 gene expressions in RAW 264.7 macrophages. Vascul Pharmacol, 2007. 47(2-3): p. 99-107.
43.Chen, H.H., et al., Inhibition of cyclic strain-induced endothelin-1 secretion by baicalein in human umbilical vein endothelial cells. Planta Med, 2006. 72(14): p. 1318-21.
44.Gruber, R., et al., Platelet-released supernatants increase migration and proliferation, and decrease osteogenic differentiation of bone marrow-derived mesenchymal progenitor cells under in vitro conditions. Platelets, 2004. 15(1): p. 29-35.
45.Werle, M., et al., MCP-1 induces activation of MAP-kinases ERK, JNK and p38 MAPK in human endothelial cells. Cardiovasc Res, 2002. 56(2): p. 284-92.
46.Shono, T., H. Kanetake, and S. Kanda, The role of mitogen-activated protein kinase activation within focal adhesions in chemotaxis toward FGF-2 by murine brain capillary endothelial cells. Exp Cell Res, 2001. 264(2): p. 275-83.
47.Liang, K.W., et al., Berberine inhibits platelet-derived growth factor-induced growth and migration partly through an AMPK-dependent pathway in vascular smooth muscle cells. Eur J Pharmacol, 2008.
48.Lanzetti, L., Actin in membrane trafficking. Curr Opin Cell Biol, 2007. 19(4): p. 453-8.
49.Honore, S., E. Pasquier, and D. Braguer, Understanding microtubule dynamics for improved cancer therapy. Cell Mol Life Sci, 2005. 62(24): p. 3039-56.
50.Weis, S.M., Evaluating integrin function in models of angiogenesis and vascular permeability. Methods Enzymol, 2007. 426: p. 505-28.
51.Lyons, A.J. and J. Jones, Cell adhesion molecules, the extracellular matrix and oral squamous carcinoma. Int J Oral Maxillofac Surg, 2007. 36(8): p. 671-9.
52.http://baike.baidu.com/view/271319.htm.