- 學位論文
Baicalein, KS-5與YD-3對血管平滑肌及內皮細胞之生長調控作用及在血管再阻塞與血管新生之藥效評估
Effects of Baicalein, KS-5 and YD-3 on Growth Regulation in Vascular Smooth Muscle Cells and Endothelial Cells, and Therapeutic Evaluation in Models of Restenosis and Angiogenesis
摘要
根據行政院衛生署公佈台灣地區九十五年度的十大死因中，惡性腫瘤排名第一，腦、心血管疾病分別佔第二及第三位，而這些疾病都與血管病變有關。血管主要由內層的內皮細胞和外側的平滑肌細胞所構成，平滑肌細胞的增生是血管再阻塞主因，也造成動脈粥狀硬化治療的瓶頸；而內皮細胞的增生，則是血管新生的必要步驟，控制血管新生對於治療發炎、風濕性關節炎甚至腫瘤的生長和轉移都有莫大的助益。因此，本論文分別探討對於血管平滑肌及血管內皮細胞增生具有抑制作用的藥物及研究其機轉。 第一部分討論中藥黃芩的主成分baicalein在體內及體外實驗中抑制血管平滑肌細胞的增生。實驗結果發現，baicalein對血清刺激造成的平滑肌細胞增生具有抑制作用，其機轉是經由抑制ERK1/2和Akt蛋白的磷酸化，影響Ras/Raf/MEK/ERK和PI-3K/Akt訊息傳遞路徑，同時也抑制IκB和DNA結合的能力。另外也減少cyclin D1的表現，使細胞週期無法進入S/G2/M期。在體內動物實驗中，口服baicalein可以有效抑制大鼠頸動脈氣球擴張損傷後的血管再阻塞。 第二部份探討acridone衍生物KS-5對血管新生促進因子bFGF引起血管內皮細胞增生的抑制作用及機轉。細胞增生 (cell proliferation) 要經過細胞生長 (cell growth) 使體積增加，和細胞分裂 (cell division) 使數目增多，過程中需要蛋白質和DNA合成。KS-5呈濃度相關性的抑制bFGF活化的ERK1/2和Akt訊息傳遞路徑並抑制細胞的DNA合成；同時KS-5也會抑制bFGF活化的mammalian target of rapamycin (mTOR)，以及mTOR下游主要的因子eIF4E和p70S6K進而抑制細胞的蛋白質合成，因此有效的抑制了bFGF造成的內皮細胞增生。利用Matrigel的實驗也證明KS-5在動物體內確實具有活性，可以明顯抑制bFGF造成的血管新生。 第三部份探討indazole衍生物YD-3抑制thrombin造成血管新生的作用和機轉。YD-3在細胞實驗中可以專一性的抑制thrombin造成的內皮細胞增生，對於PAR-1和PAR-4之activating peptides (APs) 也同樣具有作用，但對PAR-2 AP造成的細胞增生則沒有明顯的抑制作用。在體內動物實驗中也發現，不論是thrombin、PAR-1或PAR-4 AP造成的血管新生，YD-3都能有效抑制，卻不影響VEGF引起的血管新生作用。YD-3的抑制作用並不是透過抑制ERK1/2的磷酸化，而是和抑制thrombin造成的細胞Flk-1表現增加有關。 經由以上實驗結果認為baicalein、KS-5和YD-3可以抑制血管平滑肌細胞或是血管內皮細胞的增生，並且也在動物體內實驗中證明具有活性，因此很有潛力繼續研究發展成為預防血管再阻塞或是血管新生相關疾病的治療藥物。
並列摘要
Arterial reconstruction procedures, including balloon angioplasty, stenting and coronary artery bypass, are used to restore blood flow in atherosclerotic arteries. Restenosis of these arteries is a major limitation of the application of these procedures. Post-angioplasty restenosis results from two major processes: neointimal formation and constrictive remodelling. Neointimal formation is initiated by arterial injury with a resultant loss of contractile phenotype in tunica media, leading to VSMC migration and proliferation. In this dissertation, we study the effects of some biologically active chemical compounds on the proliferation of VSMC and endothelail cells and trying to elucidate their action mechanisms. In the first chapter, we investigate baicalein-mediated inhibitory effects on VSMCs proliferation and intimal hyperplasia after balloon angioplasty in the rat. Baicalein significantly inhibits serum-induced cell proliferation via decreasing the phosphorylation of ERK1/2 and Akt proteins. Baicalein inhibits cyclin D1 expression resulting in the blockade of cell cycle progression. Furthermore, bacalein attenuates serum-induced DNA binding activity of NF-κB. It also inhibits intimal hyperplasia after balloon vascular injury in rat, implying its therapeutic potential for treating restenosis after angioplasty. Angiogenesis, the process of new blood vessel formation from pre-existing ones, plays a key role in various physiological and pathological conditions, including embryonic development, wound repair, inflammation, and tumor growth. Moreover, proliferating endothelial cells undergoing DNA synthesis are a common hallmark of angiogenic microvascular spouts. In the next two chapters, we investigate the effects of KS-5 and YD-3 on bFGF- and thrombin-induced angiogenesis in cultured human umbilical vein endothelial cells (HUVECs) in vitro and in vivo. KS-5, an acridone-derivative compound, inhibits bFGF-induced cell proliferation in a concentration-dependent manner without exhibiting any significant cytotoxicity. The inhibitory effect is associated with decreasing DNA synthesis and abrogating ERK1/2 and Akt protein phosphorylation in endothelial cells. In addition, KS-5 also inhibits bFGF-induced phosphorylation of mTOR and the major downstream effectors, eIF4E and p70S6K, and leading to decreasing protein synthesis. Most importantly, KS-5 treatment in nude mice inhibited in vivo angiogenesis as revealed by matrigel implant assay. YD-3 [1-benzyl-3(ethoxycarbonylphenyl)-indazole], a selective thrombin inhibitor, inhibits neovascularization in vivo induced by thrombin, protease-activated receptor (PAR) -1, and PAR-4, but not by vascular endothelial growth factor (VEGF). YD-3 also inhibits thrombin-, PAR-1- and PAR-4-, but not PAR-2-induced cell proliferation. YD-3 predominantly inhibits thrombin-induced VEGF receptor 2 (Flk-1) up-regulation, but not phosphorylation of ERK1/2 protein. Moreover, in a murine xenograft tumor model, YD-3 administered orally reveals significant antitumor activity without cytotoxicity. In conclusion, the present study suggests that baicalein, KS-5 and YD-3 have antiproliferative effects both in vitro and in vivo and worthy of further development into drug candidates for treating restenosis and angiogenesis-dependent diseases.
參考文獻
延伸閱讀
- 林金潭（2013）。比較血管收縮素轉換酵素抑制劑與血管張力素受體阻斷劑控制高血壓病患併發其他疾病機率及醫療資源利用之相關性探討〔碩士論文，高雄醫學大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0011-1507201311155800
- 陳奕璋（2012）。血管內皮生長因子及其受器等之表現、純化及利用小角度散射進行結構分析〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU.2012.00760
- Chen, Y. F. (2018). 血管新生與標靶藥物於腫瘤治療之應用:磁振造影灌流與擴散影像之功能性評估及血管內皮細胞存活之訊息傳導 [doctoral dissertation, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU201800322
- 侯信安（2007）。Expression of angiopoietins and vascular endothelial growth factors on bone marrow mononuclear cells from acute myeloid leukemia patients and their clinical significance〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU.2007.01313
- Shi, Y. (2006). Endothelial dysfunction and changes in vascular smooth muscle responsiveness in femoral arteries of rats with type I diabetes [doctoral dissertation, The University of Hong Kong]. Airiti Library. https://www.airitilibrary.com/Article/Detail?DocID=U0029-1812201200014220