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研究生: 魏瑋昕
Wei-shin Wei
論文名稱: 藉由微流道系統量測細胞於帶電表面之貼附力
Measurement of Cell Adhesive Force on Charged Surface by Multiple-Channel Microfluidic Device
指導教授: 阮若屈
Ruoh-Chyu, Ruaan
口試委員:
學位類別: 碩士
Master
系所名稱: 生醫理工學院 - 生物醫學工程研究所
Graduate Institute of Biomedical Engineering
論文出版年: 2013
畢業學年度: 101
語文別: 中文
論文頁數: 104
中文關鍵詞: 聚二甲基矽氧烷微流道雙離子性大鼠骨髓間葉幹細胞剪應力
外文關鍵詞: Polydimethylsiloxane, PDMS, microfluidic, zwitterionic, Sprague-Dawley rat bone marrow stem cells, shear stress
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  • 本研究主要以剪應力計算細胞與不同材料表面間的貼附能力,進一步提出細胞純化的指標方法。首先,我們合成一系列雙性高分子,其親水部分由完全攜帶負電荷,負電荷與雙離子(zwitterionic)混合,或全然為雙離子的結構所組成。我們將二甲氨基丙胺(3-(Dimethylamino)-1-propylamine, DMAPA)與乙胺(Ethylamine, EA)以不同比例混合,然後與聚2,5-呋喃二酮-1-十八烯(Poly(maleic anhydride-alt-1-octadecene), MAO)上的maleic anhydride進行開環,可形成一具有長碳鏈疏水片段,帶羧酸基及雙離子性結構的共聚高分子。此高分子一方面可藉由其疏水片段吸附於聚二甲基矽氧烷(Polydimethylsiloxane, PDMS)基材,另一方面亦可藉由曝露在外的負電荷與雙離子性官能基來改變細胞貼附力。我們選用的細胞為大鼠骨髓間葉幹細胞(Sprague-Dawley rat bone marrow stem cells, RM )、小鼠胚胎纖維母細胞 (Mouse embryonic fibroblast cell line, NIH-3T3)與人類纖維腫瘤(HT1080)。細胞經12小時培養後,發現三種細胞於PDMS表面上的細胞型態多為梭長及多角型,而在親水部分全為雙離子結構的MAO-DMAPA-EA1:8:0表面,則多為圓型。當反應時EA比例逐漸增加,也就是負電荷逐漸增加時,細胞貼附型態由圓型轉為梭長或多角型,且細胞貼附量也隨EA比例增加而增多。進一步,我們將所合成之共聚高分子塗佈於微流道內,改變流速,進行細胞貼附力的量化測試,可以得知不同細胞在不同表面的附著力,從而設計出細胞純化篩選的方法。


    In this study, we coated the amphipathic charged polymers on polydimethylsiloxane (PDMS) surface for cell adhesion. To examine the cell adhesion force, the multiple-channel microfluidic device was used. The ring opening reaction between poly(maleic anhydride-alt- 1-octadecene (MAO) and different molar ratio of ethylamine (EA) and 3-(Dimethylamino)-1-propylamine (DMAPA) were carried out. From the analysis of NMR spectrum, we obtain the MAO:DMAPA=1:8 with all maleic anhydride functional groups being 100 % ring opening and this polymer possesses zwitterionic functional group in co-polymer at pH 7.4. For MAO-DMAPA-EA co-polymer synthesis, we found the content of zwitterionic functional groups would decrease with increasing the addition of EA, and the negatively charged groups would also increase in co-polymer. After coating the co-polymers with different charged group content on PDMS surface, the hydrophilicity of surface and anti-protein adsorption ability would increase as the zwitterionic functional groups increase on co-polymer. Furthermore, the primary cell sprague-dawley rat bone marrow stem cells (RM), mouse embryonic fibroblast cell line (NIH-3T3), and human sarcoma cell line (HT1080) were cultured on co-polymer coated surface for 12 hr. We found that the morphology of adhesive NIH-3T3 cells was spindle-like and polygonal on PDMS surface and spherical shape on MAO-DMAPA-EA 1:8:0 coated surface. With the increase of EA content on co-polymer, the morphology of cellular adhesion turned from spherical into spindle-like and polygonal. The amount of cellular adhesion increased with the ethylamine composition increase. Similar results were also observed on RM and NIH3T3 cells adhesion. We obtain the shear forces of RM on the co-polymer surface are ranging from 0 to 4.62 dyne/cm2. The shear stress of cell adhesion is related to the zwittionic content on co-polymer, that is, the high zwitterionic content would result in low adhesion force.

    目錄 摘要 I Abstract III 圖目錄 VIII 表目錄 XI 第一章 緒論 1 1.1研究動機 1 1.2研究目的 2 第二章 文獻回顧 4 2.1 細胞類型及貼附機制 4 2.1.1細胞類型 4 2.1.2 細胞貼附機制 4 2.2 細胞分選 8 2.2.1常見的細胞分選方式 10 2.3微流體晶片之簡介 17 2.3.1微流道之材料 18 2.3.4 PDMS Microchip 的表面修飾方法 19 2.4生物分子與材料表面之交互作用 23 2.4.1 蛋白質與材料表面作用之機制 23 2.4.2雙離子性高分子 25 第三章 實驗藥品、儀器設備與方法 30 3.1實驗藥品及儀器設備30 3.1.1兩性雙離子性高分子製備及PDMS表面改質 30 3.1.2 微流體晶片製程及實驗儀器 32 3.1.3 細胞培養 33 3.2 實驗方法 34 3.2.1實驗架構 34 3.2.2 兩性雙離子性高分子之製備及性質鑑定 35 3.2.3 PDMS表面改質方法及表面性質測定 37 3.2.4 微流體晶片製程 39 3.2.5細胞培養 47 第四章 結果與討論 52 4.1兩性雙離子性共聚高分子之合成 52 4.1.1 MAO-DMAPA-EA高分子之合成 52 4.2 兩性雙離子性高分子之結構與物化特性 54 4.2.1 MAO-DMAPA-EA高分子之結構鑑定 54 4.3 MAO-DMAPA-EA之物化性質 58 4.3.1 MAO-DMAPA-EA之pKa之量測 58 4.3.2 MAO-DMAPA-EA之溶解性 60 4.4 MAO-DMAPA-EA在PDMS表面之塗佈 61 4.4.1量測MAO-DMAPA-EA塗佈表面之親疏水性 61 4.4.2高分子塗佈於PDMS基材表面之元素分析 67 4.4.3蛋白質於高分子塗佈表面的吸附行為 68 4.5 細胞於MAO-DMAPA-EA塗佈表面之貼附行為 70 4.6 流場剪應力對細胞貼附力之影響 75 4.6.1晶片內各培養區的細胞均勻度 75 4.6.2微流道與流場剪應力之關係 77 第五章 結論 85 參考文獻 87

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