2012-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/650662摘要：動脈硬化及氣球擴張術後血管再狹窄的形成過程中，血管組成細胞—內皮細胞和平滑肌細胞均扮演很重要的角色，其特徵為內皮細胞失去功能，造成相關蛋白產生並且造成平滑肌細胞由血管中層移行至血管內層並在血管內層大量增生，由這些現象顯示內皮細胞與平滑肌細胞會交互作用與影響。氧化壓力對於動脈硬化及血管再狹窄的發生及進展具有密切的相關性，因此全盤深入瞭解在中藥上具有”延年益壽”功效的抗氧化及抗發炎試劑—靈芝多醣體對動脈硬化與血管再狹窄形成之影響及影響機制是一重要的課題。第一年計畫本實驗室預定以內皮細胞和平滑肌細胞共同培養，接受剪力處理，盡量模擬成生物體內的血管模式，檢測以靈芝多醣體處理內皮細胞是否能夠影響在細胞受到發炎因子(TNF-α)刺激下產生的活性氧族群與凝血酶調節素，而達到保護細胞的效果。進一步使用西方墨漬法與EMSA分析法研究PI3K-Akt，細胞分裂素活化蛋白激酶，與轉錄因子是否涉及這些相關蛋白的產生。另外也檢測靈芝多醣體是否會影響凝血酶調節素mRNA的穩定度及其相關機轉。第二年計畫以靈芝多醣體處理與內皮細胞共同培養的平滑肌細胞是否能夠影響細胞受到TNF-α刺激下所產生的活性氧族群、合成相關蛋白、移行與增生，並探討其機轉。我們會先檢視TNF-α對於平滑肌細胞進行移行與增生的作用，並觀察靈芝多醣體是否透過減少活性氧族群、分解間質的細胞間質蛋白酶(MMP-2及MMP-9)而影響TNF-α對細胞產生的移行作用。另外，使用西方墨漬法檢驗靈芝多醣體影響細胞表現凝血酶調節素與細胞分裂有關蛋白。進一步研究靈芝多醣體是否亦經由PI3K-Akt，細胞分裂素活化蛋白激酶與轉錄因子的訊息傳遞路徑影響細胞的相關蛋白產生、移行與增生。第三年計畫，預計使用餵食膽固醇飼料的apo-E 缺乏小鼠所產生的動脈硬化與餵食膽固醇飼料並進行去內皮手術的apo-E缺乏小鼠的新內膜形成，以靈芝多醣體處理，觀察是否會影響內膜面積與細胞增生。本計畫亦將以第一年、第二年的實驗結果，檢測靈芝多醣體影響凝血酶調節素、MMPs在分支或未分支處之血管壁的表現，同時利用辨識內皮細胞、平滑肌細胞與巨噬細胞的特定抗體，以免疫組織化學染色法分析蛋白表現強度與血管分支處及細胞種類的相關性。此三年結果可詳細闡明血管細胞共同培養及接受剪力處理之模式的重要性與靈芝多醣體在動脈硬化或血管再狹窄中所扮演的角色、功能及其調控機制，此研究成果將對今後動脈硬化或血管再狹窄的治療與預防有很大助益。<br> Abstract: The endothelial cells (ECs) and smooth muscle cells (SMCs) play the important roles in thepathogenesis of atherosclerosis and of restenosis following balloon angioplasty. In both phenomena,the characteristic changes are the dysfunction of ECs and the migration of SMCs from the media andtheir proliferation within the intima. The development and clinical sequelae of atherosclerosis andrestenosisare are also closely associated with oxidative stress and inflammation. Reishi (Ganodermalucidum) polysaccharides have long been known as health foods and used as traditional Chinesemedicines and the anti-inflammatory, antioxidant, and immunomodulatory actions are thepharmacological properties proposed to underlie their beneficial effects. Consequently, fullunderstanding of the role of Reishi polysaccharides, which is involved in the mechanism ofatherosclerotic and restenotic development, is critically important. In the present study, a coculture flowsystem is developed to study the effect of Reishi polysaccharides and inflammatory cytokines (TNFα)on EC-SMC interactions. ECs and SMCs were separated by a porous membrane with only the EC sidesubjected to the flow condition. The goals of the project in the 1st year, therefore, are to investigatewhether the treatment of Reishi polysaccharides on ECs in coculture with SMCs can affect theexpression of thrombomodulin under TNFα stimulation. Furthermore, the cells with Reishipolysaccharides treatment will be subjected to western blot analysis and electrophoretic mobility shiftassays to investigate whether phosphoinositide 3-kinase (PI3K)-Akt, mitogen-activated protein kinases(C-Jun N-terminal kinase, extracellular signal–regulated kinase, p38), and transcriptional factors(Nuclear factor-B and activator protein-1) involved in the signaling pathway leading to the productionof the related proteins. The goals of the project, in the 2nd year, are to investigate whether the treatmentof Reishi polysaccharides for SMCs by coculture with ECs can affect the expression ofthrombomodulin, cell migration, and cell proliferation under TNFαstimulation. We will examine thesuperoxide anions and hydrogen peroxide production involved in the migration and proliferation ofSMCs. We will seek to determine whether matrix metalloproteinase-2 (MMP-2) and matrixmetalloproteinase-9 (MMP-9), which can decompose matrix, are critical for the migration ofTNFα-treated SMCs. To test the ability of Reishi polysaccharides to modulate the expression of MMPsand cell cycle-related proteins, gelatin zymography and western blot analysis will be used, respectively.Furthermore, the cells with Reishi polysaccharides treatment will be subjected to western blot analysisand electrophoretic mobility shift assays to investigate whether PI3K-Akt, MAPKs, and transcriptionalfactors involved in the signaling pathway leading to protein production, cell migration and proliferation.In addition, we will study the effects of Reishi polysaccharide treatment on atherosclerotic lesions ofcholesterol-fed apo-E deficient mice and on the neointimal formation of endothelial-denudedcholesterol-fed apo-E deficient mice in the 3rd year. According to the results of the 1st and 2nd years inTNFα-treated vascular cells, we will also examine the effects of Reishi polysaccharides onthrombomodulin and MMPs expressions in arterial walls of apo-E deficient mice. Antibodies specificfor endothelial cells, smooth muscle cells, and macrophages will be applied to distinguish different celltypes by immunohistochemistry, in order to correlate protein expression with cell types. Our findingswill elucidate that the coculture flow system could be useful in better understanding EC/SMCcross-talk and the functional consequence of their interactions. In addition, these results will provide amore through understanding of the protective role of the Reishi polysaccharides in atherosclerosis andrestenosis剪應力內皮細胞平滑肌細胞凝血?調節素動脈硬化血管再狹窄shear stressendothelial cellsmooth muscle cellsthrombomodulinatherosclerosisrestenosis.