| 摘要: | 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-?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. |
