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研究生:王慧英
研究生(外文):Wang HI
論文名稱:黃芩苷元對內皮細胞之附著﹐移動及架構蛋白分佈之影響
論文名稱(外文):Effects of Baicalein on Adhesion, Migration andSubcellular distribution of Cytoskeletal Proteins in Rat Heart Endothelia Cells
指導教授:葛其梅葛其梅引用關係徐士蘭徐士蘭引用關係
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學院碩士在職專班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:60
中文關鍵詞:黃芩苷移動附著架構蛋白
外文關鍵詞:BaicaleinMigrationAdhesionCytoskeletal protein
相關次數:
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中文摘要
過去本實驗室發現黃芩苷元(Baicalein)處理後的心臟內皮細
胞,其生長受抑制,且其細胞形態變化明顯,細胞外形變大變薄。
由前人的文獻中,我們知道細胞的形態變化受到細胞內架構蛋白分
佈組成之影響,且細胞形態的變化可改變細胞的附著及移動能力。
本研究的目的在探討黃芩苷元作用下細胞內重要架構蛋白在細胞內
之分佈及表現量,並進一步探討黃芩苷元是否也影響內皮細胞的移
動及附著的能力。我們取出生四天的大白鼠 幼鼠,取其心臟分離培
養心臟內皮細胞作為實驗的內皮細胞模式,以100 μΜ黃芩苷元處
理培養第二或第三代的內皮細胞,加藥作用四天後,以細胞附著實
驗、移動能力測定、細胞免疫螢光分析及西方墨點法等技術,分析
黃芩苷元對心臟內皮細胞的附著作用、移動作用及架構蛋白表現及
分佈之影響。結果發現黃芩苷元處理抑制了內皮細胞的移動能力但
卻增強了附著作用。由免疫螢光分析及西方墨點法的實驗結果我們
發現,在黃芩苷元處理的內皮細胞中,參與細胞移動作用的架構蛋
白actin及tubulin在細胞內的表現量及分佈明顯減少。而附著作
用分析的結果顯示,經黃芩苷元處理後,內皮細胞的附著能力明顯
增強。進一步探討處理黃芩苷元之內皮細胞究竟與何種胞外間質蛋
白(extracellular matrix proteins)之間的作用會增強。我們以
collagen﹐laminin﹐fibronectin及vitronectin處理培養盤底
後,再測試內皮細胞與這四種分子間之附著能力。結果發現黃芩苷
元處理後之內皮細胞與fibronectin及vitronectin兩細胞外間質
蛋白質間之附著能力增強,但卻collagen與及laminin兩分子間
之附著能力明顯減弱。以西方墨點法分析與細胞附著作用有關的焦
點附著蛋白(focal adhesive protein) paxillin及vinculin 分
子表現量也有增加的趨勢。由以上的實驗結果我們推測黃芩苷元抑
制內皮細胞的移動作用,可能是因黃芩苷元改變了內皮細胞內骨架
蛋白中之肌動蛋白(Actin)及微管蛋白(Tubulin)的結構,並抑制此
兩蛋白質在內皮細胞內的表現量。而黃芩苷元促進內皮細胞內的附
著能力,可能是因黃芩苷元造成細胞的焦點附著區(Focal dhesion
plaque)的穩定而促使細胞與胞外間質之間的附著作用增強之原因

Abstract
Our previous studies demonstrated that baicalein can inhibit
cell proliferation and change the morphology of the rat heart endothelial cells (RHEC).The baicalein-treated RHEC were more
flatter and larger than control untreated cells.The effect of cytoskeleton reorganization affects cell shape that can modulate the adhesion and migration of cells. In this study﹐we investigated the subcellular distribution of cytoskeletal proteins﹐and examined the effect of baicalein on the adhesion and migration of RHEC. The rat heart endothelia cells were isolated from the four-days-old rat and the secondary sub-
cultured cells were treated with 100 μM baicalein for four days. Cell migration assay﹐adhesion assay﹐double-label immuno-
fluorescence staining and western blot analysis were performed. Our results indicated that baicalein inhibited the migration
and enhanced the adhesion of RHEC to extracellular matrix. We also found that the decrease of actin and tubulin filaments of baicalein treated cells were detected by immunofluorescence staining and western blot. The Focal adhesive proteins﹐ paxillin and vinculin proteins, releated to the cell adhesion, increased in baicalein-treated RHEC.Based on our results, we suggest that baicalein-mediated migration inhibition might be
due to the reason why baicalein can inhibit the migration of cell changes of the cytoskeletal structures of actin and tubulin the cell. Increase in the expression of paxillin and vinculin might be contributed to the baicalein-increased adhesion.
目錄
目錄……………………………………………………………………Ι
中文摘要………………………………………………………………Ⅳ
英文摘要………………………………………………………………Ⅵ
壹. 前言……………………………………………………………….1
貳. 材料與方法……………………………………………………….12
一. 試劑種類及來源………………………………………………12
二. 細胞株來源……………………………………………………13
三. 藥劑之配置與細胞的培養…………………………………..14
1.DMEM細胞培養液之配置……………………….………….14
2.完全培養液…………………………………………………14
3.黃芩苷元藥劑……………………………………………..14
4.內皮細胞培養法……………………………………….….14
四. 細胞移動能力之分析…………………………………………15
五. 內皮細胞在不同胞外間質之吸附能力試驗…………………16
六. 免疫螢光染色…………………………………………………17
1. 細胞之培養與固定……………………………………….17
2. 細胞染色…………………………………………………...17
七. 蛋白質分析…………………………………………………..18
1. 收取細胞…………………………………………………18
2. 蛋白質之萃取……………………………………………18
3. 聚丙烯醯胺膠體電泳法…………………………………19
4. 西方墨點法………………………………………………20
5. 統計方法…………………………………………………21
參. 結果………………………………………………………………22
(一) 黃芩苷元抑制內皮細胞移動作用………………………22
(二) 黃芩苷元增加內皮細胞附著的能力……………………22
(三) 黃芩苷元對在內皮細胞架構蛋白在細胞內分佈之影響.24
1.黃芩苷元對actin在內皮細胞內分佈之影響……………24
2.黃芩苷元對tubulin在內皮細胞內分佈之影響…………25
3.黃芩苷元對vimentin在內皮細胞內分佈之影響……….25
4.黃芩苷元對vinculin在內皮細胞內分佈之影響……….26
5.黃芩苷元對paxillin在內皮細胞內分佈之影響……….26
6.黃芩苷元對talin在內皮細胞內分佈之影響……………27
肆. 討論……………………………………………………… 28
一. 黃芩苷元抑制架構蛋白在細胞中移動的探討………….28
二. 黃芩苷元促進架構蛋白在細胞中附著的探討………….32
伍. 參考文獻………………………………………………………..36
陸. 圖……………..,…………………………………………………43
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