Formosa Fabio, Anfuso Carmelina D, Satriano Cristina, Lupo Gabriella, Giurdanella Giovanni, Ragusa Nicola, Marletta Giovanni, Alberghina Mario
Laboratory for Molecular Surfaces and Nanotechnology, Department of Chemical Sciences, University of Catania and CSGI, 95125 Catania, Italy.
Microvasc Res. 2008 Apr;75(3):330-42. doi: 10.1016/j.mvr.2007.11.005. Epub 2007 Dec 4.
We examined the adhesion and proliferation of immortalized endothelial cells GP8.39 (ECs) onto polyethyleneterephtalate (PET) and polyhydroxymethylsiloxane (PHMS) thin films, functionalized by UV-O(3) treatment and/or protein immobilization. The modified surface topography showed partial oxidation for both polymers, a slight increase in wettability and monopolar basic character for PET, and a hydrophilic bipolar acid-base behaviour for PHMS. UV-O(3) treatment did not induce significant roughness changes (under 1 nm) as shown by atomic force spectroscopy measurements (AFM). The EC adhesion and spreading onto untreated and modified surfaces were investigated both before and after immobilization of collagen (CA) and fibronectin (FN) adlayers. AFM analyses showed an open-weave protein layer on both untreated polymers which became a tight-woven net after UV-O(3) irradiation of underlying films. On day 5 after seeding, cell count analyses on irradiated PET surfaces, CA/FN-coated or not, showed EC adhesion and proliferation significantly greater than those on untreated polymers, indicating that UV-O(3) irradiation promoted fast endothelialization. A less pronounced EC spreading behaviour on treated PHMS was observed. In ECs grown on irradiated and CA- or FN-coated PET, the levels of phospho-protein kinase Calpha (p-PKCalpha, phospho-ERK1/2, and phospho-cytosolic phospholipase A(2) (p-cPLA(2)), all enzymes taken as signaling markers of cell adhesion and proliferation, decreased in comparison to those in CA- or FN-coated untreated PET. In contrast, in ECs grown on UV-O(3)-treated PHMS, Western blot analyses showed increased levels of p-PKCalpha, p-ERK1/2 and p-cPLA(2) in comparison with cells grown onto untreated polymer. The growth response of ECs to the substrates was related to the changes of polarity properties of UV-O(3)-treated polymer films, from hydrophobic/neutral towards hydrophilic/charged layers, and the signaling pathway remodelling to the cell proliferation degree.
我们研究了永生化内皮细胞GP8.39（ECs）在经紫外线臭氧（UV - O(3)）处理和/或蛋白质固定化功能化的聚对苯二甲酸乙二酯（PET）和聚羟甲基硅氧烷（PHMS）薄膜上的黏附与增殖情况。改性后的表面形貌显示两种聚合物均发生了部分氧化，PET的润湿性略有增加且具有单极性碱性特征，而PHMS具有亲水性双极性酸碱行为。原子力光谱测量（AFM）表明，UV - O(3)处理未引起显著的粗糙度变化（小于1纳米）。在固定胶原蛋白（CA）和纤连蛋白（FN）吸附层之前和之后，均对ECs在未处理和改性表面上的黏附与铺展情况进行了研究。AFM分析显示，在两种未处理的聚合物上均有开放编织的蛋白质层，在对下层薄膜进行UV - O(3)辐照后，该蛋白质层变成了紧密编织的网络。接种后第5天，对辐照后的PET表面（无论是否涂覆CA/FN）进行细胞计数分析，结果显示ECs的黏附与增殖显著高于未处理的聚合物，这表明UV - O(3)辐照促进了快速内皮化。在处理过的PHMS上观察到ECs的铺展行为不太明显。在辐照且涂覆CA或FN的PET上生长的ECs中，与涂覆CA或FN的未处理PET相比，磷酸化蛋白激酶Cα（p - PKCalpha）、磷酸化细胞外信号调节激酶1/2（phospho - ERK1/2）和磷酸化胞质磷脂酶A2（p - cPLA(2)）（所有这些酶均被视为细胞黏附与增殖的信号标志物）的水平均降低。相反，在UV - O(3)处理的PHMS上生长的ECs中，蛋白质印迹分析显示，与在未处理聚合物上生长的细胞相比，p - PKCalpha、p - ERK1/2和p - cPLA(2)的水平升高。ECs对底物的生长反应与UV - O(3)处理的聚合物薄膜极性性质的变化有关，即从疏水/中性变为亲水/带电层，以及信号通路重塑与细胞增殖程度的关系。
