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题名: 膠原蛋白與明膠電紡絲之基材剛性對於類骨母細胞 MG63 細胞活性之影響
The difference stiffness of matrix cause MG63 osteoblast-like cell exhibit different behavior when grown on electrospun collagen matrix versus electrospun gelatin matrix
作者: Hau-min Liou
劉灝泯
贡献者: NTOU:Institute of Bioscience and Biotechnology
國立臺灣海洋大學:生物科技研究所
关键词: 膠原蛋白;明膠;電氣紡絲;剛性
Collagen;Gelatin;Electrospun;Stiffness
日期: 2011
上传时间: 2011-11-25T07:36:21Z
摘要: 電氣紡絲為一種方便與有效率製備胞外基材的技術。但是在電氣紡絲的製備過程中所使用的 fluorinated alcohol 溶劑,會使得蛋白質變性以及喪失其原有的特殊構型。藉由穿透式電子顯微鏡與 circular dichroism 分析結果得知,將膠原蛋白溶於六氟異丙醇 (HFIP) 中,會使得膠原蛋白喪失其原有 triple helix 結構,使其構型與明膠相似。然而,在基材機械力學的分析中,我們發現膠原蛋白與明膠基材的楊氏模量 (Young modulus) 分別為 94.29 ± 15.18 與 71.88 ± 21.10 MPa,而抗拉強度(ultimate tensile strength)分別為 1.93 ± 0.37 and 0.93 ± 0.46 MPa。故本研究將探討當類骨母細胞 MG63 培養於擁有不同的機械力學特性的膠原蛋白電紡絲基材與明膠電紡基材是否會對與類骨母細胞 MG63 貼附、增生、成骨基因與蛋白質的分泌產生不同的表現進行探討分析。由貼附與增生實驗得知,將類骨母細胞 MG63 培養於膠原蛋白與明膠電紡絲基材上,並沒有明顯的差異。在基因表現上,我們可以發現類骨母細胞 MG63 於膠原蛋白電紡絲基材上,其 OPN、type I collagen、ALP 與 OCN 的基因表現量都相較於培養於明膠電紡絲基材上高。除此之外,藉由西方點墨法,我們可以發現將類骨母細胞 MG63 培養於膠原蛋白電紡絲基材上,其 Y397-FAK、 S910-FAK、ERK1/2、 BSP 與 OPN 蛋白質表現量都相較於培養於明膠電紡絲基材上高。由上述實驗所知,我們推測因為膠原蛋白電紡絲相較於明膠電紡絲擁有較良好的機械力學強度,導致類骨母細胞MG63 培養於膠原蛋白電紡絲時有較高的 focal adhesion 表現,進而提高骨分化相關因子之表現量。
Electrospinning technique is a convenient and efficient method of fabricating nanofiber extracellular matrix (ECM). However, using the fluorinated alcohol solvent in electrospinning fabrication process will cause the protein denaturation and losing the specific charactics. From transmission electron microscopy (TEM) and circular dichroism analysis indicated the collagen dissolving in the hexafluoroisopropanol(HFIP) will lose their original triple helix structure and becoming the random coil structure similar to the gelatin. However, the Young's modulus of the collagen and gelatin matrices were 94.29 ± 15.18 and 71.88 ± 21.10 MPa, respectively, and their ultimate tensile stresses were 1.93 ± 0.37 and 0.93 ± 0.46 MPa,respectively. In this study, we will examine the adhesion, proliferation, osteogenesis-related gene expression and related protein secretion in MG63 osteoblast-like cells that were cultured on the two matrices. From the cell adhesion and proliferation assay we found that there were no significantly differences on these two matrices. However, the gene expression levels of OPN, type I collagen, ALP, and OCN were significantly higher in MG63 osteoblast-like cells grown on the electrospun collagen matrix than in those grown on the electrospun gelatin matrix. In addition, the western blot assay also finding the protein expression of Y397-FAK, S910-FAK, ERK1/2, BSP, and OPN proteins were also higher expression level on the electrospun collagen matrix than on the electrospun gelatin matrix. Above all of date we examine, we speculate that the mechanical properties of the electrospun collagen matrix may induce the osteogenic differentiation ability than electrospun gelatin matrix.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M98360005
http://ntour.ntou.edu.tw/handle/987654321/30198
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