钛表面肝素和纤维连接蛋白微图形的构建及其对血小板和内皮细胞行为的影响
发布时间：2018-10-12 11:57
【摘要】：对心血管人工植入材料而言,内膜增生和血栓的形成是其植入后面临的主要问题。通过表面改性技术促进内皮细胞粘附的同时减少血小板的粘附能有效提高材料的生物相容性,其中表面图形化技术正成为研究热点。 本文采用软刻蚀技术中的微转移模塑技术在预处理的钛(Ti)表面构建仿生生物大分子微图形。钛的预处理包括NaOH碱活化和3-氨丙基三乙氧基硅烷(APTE)偶联处理,通过扫描电子显微镜(SEM)、接触角测量仪、傅里叶红外变换光谱分析(FTIR)和氨基定量的方法对预处理的钛表面进行理化表征。聚二甲基硅氧烷(PDMS)印章由浇铸法制备,运用其压印具有阻抗蛋白和细胞粘附的单甲氧基聚乙二醇琥珀酰亚胺碳酸酯(mPEG-SC),而后组装具有抗凝作用的肝素(Hep)和促进内皮细胞粘附的纤维连接蛋白(Fn)混合物(Hep-Fn),构成iPEG-SC/Hep-Fn微图形。通过SEM、接触角测量仪、FTIR、Hep及Fn的染色对制备的Hep-Fn微图形进行表征。在此基础上进行体外血小板粘附实验和内皮细胞培养实验,研究图形对血小板及内皮细胞行为的影响。 SEM、接触角、FTIR、光镜及荧光显微镜结果显示：碱活化后的钛表面变粗糙,亲水性加强,表面出现羟基的吸收峰；硅烷偶联后的钛表面粗糙度进一步加大,疏水性增大,FTIR检测有CH2的吸收峰,氨基在表面的密度约为51nmol/cm2。图形化的样品表面能看到清晰的条纹图形,FTIR出现mPEG-SC、Hep和Fn的特征吸收峰。Hep和Fn的染色进一步证实mPEG-SC的生物惰性,Hep和Fn只分布在没有mPEG-SC的图形区域。 血小板粘附实验表明：平板Ti和Hep-Fn表面血小板布满整个表面,血小板伸出伪足,没有团聚。由于肝素的抗凝作用,Hep-Fn表面血小板粘附数量少于Ti。nPEG-SC平板表面几乎没有血小板粘附；图形样品表面血小板选择性分布在Hep-Fn条纹区域,与平板的Hep-Fn相比能进一步减少血小板粘附数量。不同尺寸的图形样品Hep-Fn的有效面积不同致使血小板的粘附数量有所不同。激活的血小板在数量和分布上和粘附有着同样的趋势,图形化样品表面激活的血小板数量比平板的Ti和Hep-Fn样品少。APTT结果显示平板和图形的Hep-Fn没有明显延长APTT时间,但也没有加剧凝血时间,具有—定抗凝作用。 内皮细胞粘附结果显示：平板的Hep-Fn表面与钛相比,能促进内皮细胞的粘附、铺展、增殖；mPEG-SC表面几乎没有内皮细胞粘附。图形化表面内皮细胞都沿着Hep-Fn图形方向分布,不同尺寸的图形对内皮细胞的取向角、长宽比、形态指数等均有不同影响：大于细胞尺寸的图形使细胞呈2个或多个并行排列,有利于细胞铺展和增殖；与细胞尺寸相近的图形能够使细胞成单细胞阵列分布,拉长并产生引导作用。 以上结果表明：具有生物功能的mPEG-SC/Hep-Fn微图形能调节血小板和内皮细胞分布的同时,降低血小板粘附并调节内皮细胞粘附、生长和增殖,有效提高了钛金属的生物相容性。
[Abstract]:Intimal hyperplasia and thrombosis are the main problems after implantation of artificial cardiovascular materials. Surface modification technology can promote endothelial cell adhesion and reduce platelet adhesion, which can effectively improve the biocompatibility of materials, among which surface graphics technology is becoming a hot topic. In this paper, bionic macromolecular micrographics were constructed on the surface of pretreated titanium (Ti) by microtransfer molding in soft etching. The pretreatment of titanium includes activation of NaOH base and (APTE) coupling treatment of 3-aminopropyl triethoxy silane. The contact angle of titanium is measured by scanning electron microscope (SEM),). The surface of titanium was characterized by (FTIR) and amino quantitative analysis by Fourier transform infrared spectroscopy (FTIR). Poly (dimethylsiloxane) (PDMS) seal was prepared by casting method. After imprinting monomethoxy polyethylene glycol succinimide carbonate (mPEG-SC) with impedance protein and cell adhesion, heparin (Hep) with anticoagulant activity and fibronectin (Fn) mixture (Hep-Fn), which promoted endothelial cell adhesion, were assembled to form iPEG-SC/Hep-Fn micrograph. The Hep-Fn micrographs were characterized by SEM, contact angle measuring instrument, FTIR,Hep and Fn staining. On this basis, platelet adhesion test and endothelial cell culture were carried out in vitro. SEM, contact angle, FTIR, light microscope and fluorescence microscope showed that the surface of titanium after alkali activation became rough, hydrophilic was strengthened, and hydroxyl absorption peak appeared on the surface. After silane coupling, the surface roughness and hydrophobicity of titanium were further increased. The absorption peak of CH2 was detected by FTIR, and the density of amino on the surface was about 51 nmol / cm ~ 2. A clear pattern of stripe can be seen on the surface of a graphical sample, and the characteristic absorption peaks of mPEG-SC,Hep and Fn appear in FTIR. The staining of Hep and Fn further confirms the biological inertia of mPEG-SC. Hep and Fn are only distributed in the graphical region without mPEG-SC. Platelet adhesion test showed that platelets covered the whole surface of Ti and Hep-Fn, and platelets extended pseudopodia without agglomeration. Because of the anticoagulant effect of heparin, the number of platelet adhesion on Hep-Fn surface was less than that on Ti.nPEG-SC plate surface. Compared with plate Hep-Fn, platelet adhesion was further reduced. The number of platelet adhesion varies with the difference of the effective area of Hep-Fn in different size graphic samples. The number and distribution of activated platelets showed the same trend as that of adhesion. The number of activated platelets on the surface of graphical samples was less than that of Ti and Hep-Fn samples. The results of APTT showed that the Hep-Fn of plate and pattern did not prolong the APTT time obviously. But also did not aggravate the coagulation time, with-anticoagulant effect. The results of endothelial cell adhesion showed that the surface of Hep-Fn on the plate could promote the adhesion, spread and proliferation of endothelial cells compared with titanium, and there was almost no adhesion of endothelial cells on the surface of mPEG-SC. The morphologic surface endothelial cells are distributed along the Hep-Fn pattern. The shapes of different sizes have different effects on the orientation angle, aspect ratio and morphological index of the endothelial cells. The shapes larger than the cell size cause the cells to be arranged in two or more parallel. The pattern similar to the cell size can make the cell into a single cell array distribution, elongate and produce a leading role. The results show that the biofunctional mPEG-SC/Hep-Fn micrograph can regulate the distribution of platelet and endothelial cells, decrease platelet adhesion and regulate the adhesion, growth and proliferation of endothelial cells, and effectively improve the biocompatibility of titanium.
【学位授予单位】：西南交通大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08
本文编号：2266023
[Abstract]:Intimal hyperplasia and thrombosis are the main problems after implantation of artificial cardiovascular materials. Surface modification technology can promote endothelial cell adhesion and reduce platelet adhesion, which can effectively improve the biocompatibility of materials, among which surface graphics technology is becoming a hot topic. In this paper, bionic macromolecular micrographics were constructed on the surface of pretreated titanium (Ti) by microtransfer molding in soft etching. The pretreatment of titanium includes activation of NaOH base and (APTE) coupling treatment of 3-aminopropyl triethoxy silane. The contact angle of titanium is measured by scanning electron microscope (SEM),). The surface of titanium was characterized by (FTIR) and amino quantitative analysis by Fourier transform infrared spectroscopy (FTIR). Poly (dimethylsiloxane) (PDMS) seal was prepared by casting method. After imprinting monomethoxy polyethylene glycol succinimide carbonate (mPEG-SC) with impedance protein and cell adhesion, heparin (Hep) with anticoagulant activity and fibronectin (Fn) mixture (Hep-Fn), which promoted endothelial cell adhesion, were assembled to form iPEG-SC/Hep-Fn micrograph. The Hep-Fn micrographs were characterized by SEM, contact angle measuring instrument, FTIR,Hep and Fn staining. On this basis, platelet adhesion test and endothelial cell culture were carried out in vitro. SEM, contact angle, FTIR, light microscope and fluorescence microscope showed that the surface of titanium after alkali activation became rough, hydrophilic was strengthened, and hydroxyl absorption peak appeared on the surface. After silane coupling, the surface roughness and hydrophobicity of titanium were further increased. The absorption peak of CH2 was detected by FTIR, and the density of amino on the surface was about 51 nmol / cm ~ 2. A clear pattern of stripe can be seen on the surface of a graphical sample, and the characteristic absorption peaks of mPEG-SC,Hep and Fn appear in FTIR. The staining of Hep and Fn further confirms the biological inertia of mPEG-SC. Hep and Fn are only distributed in the graphical region without mPEG-SC. Platelet adhesion test showed that platelets covered the whole surface of Ti and Hep-Fn, and platelets extended pseudopodia without agglomeration. Because of the anticoagulant effect of heparin, the number of platelet adhesion on Hep-Fn surface was less than that on Ti.nPEG-SC plate surface. Compared with plate Hep-Fn, platelet adhesion was further reduced. The number of platelet adhesion varies with the difference of the effective area of Hep-Fn in different size graphic samples. The number and distribution of activated platelets showed the same trend as that of adhesion. The number of activated platelets on the surface of graphical samples was less than that of Ti and Hep-Fn samples. The results of APTT showed that the Hep-Fn of plate and pattern did not prolong the APTT time obviously. But also did not aggravate the coagulation time, with-anticoagulant effect. The results of endothelial cell adhesion showed that the surface of Hep-Fn on the plate could promote the adhesion, spread and proliferation of endothelial cells compared with titanium, and there was almost no adhesion of endothelial cells on the surface of mPEG-SC. The morphologic surface endothelial cells are distributed along the Hep-Fn pattern. The shapes of different sizes have different effects on the orientation angle, aspect ratio and morphological index of the endothelial cells. The shapes larger than the cell size cause the cells to be arranged in two or more parallel. The pattern similar to the cell size can make the cell into a single cell array distribution, elongate and produce a leading role. The results show that the biofunctional mPEG-SC/Hep-Fn micrograph can regulate the distribution of platelet and endothelial cells, decrease platelet adhesion and regulate the adhesion, growth and proliferation of endothelial cells, and effectively improve the biocompatibility of titanium.
【学位授予单位】：西南交通大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08
【参考文献】
相关期刊论文 前10条
1 李永刚;张平;吴一辉;宣明;;聚二甲基硅氧烷表面亲水性的研究[J];分析化学;2006年04期
2 蒋小松;陈俊英;黄楠;;APTE修饰钛氧膜并固定Ⅰ型胶原对其凝血性能的影响[J];功能材料;2008年05期
3 赵小梅,陈四海,付小潮,史锡婷,董珊,李毅,易新建;化学镀镍在32×32非致冷红外焦平面互连中的应用[J];红外技术;2005年04期
4 信思树;普朝光;杨明珠;杨培志;;一种新型的正胶剥离技术及其应用[J];红外技术;2006年02期
5 潘力佳,何平笙;纳米器件制备的新方法——微接触印刷术[J];化学通报;2000年12期
6 文学军,王小祥;金属生物材料的微粗糙表面及其生物学效应(Ⅰ)─—金属生物材料的微粗糙表面[J];生物医学工程学杂志;1997年01期
7 文学军;金属生物材料的微粗糙表面及其生物学效应(Ⅱ)──金属生物材料微粗糙表面的生物学效应[J];生物医学工程学杂志;1997年02期
8 李伯刚,那娟娟,尹光福,殷杰,郑昌琼;生物碳素材料表面血小板黏附的实验研究[J];生物医学工程学杂志;2004年01期
9 韩雪;陶晓杰;李述军;赵永康;艾红军;;新型钛合金阳极氧化后对成骨细胞增殖、分化的影响[J];实用口腔医学杂志;2006年02期
10 王奕;;软刻蚀技术[J];宿州教育学院学报;2006年05期
相关博士学位论文 前2条
1 石锦霞;软刻蚀技术在高分子科学中的应用[D];中国科学技术大学;2008年
2 孙建国;PEG水凝胶的金微图案化修饰及其表面细胞黏附行为的研究[D];复旦大学;2009年
相关硕士学位论文 前7条
1 周超;Fn在硅烷化无机材料表面固定及定量表征方法研究[D];西南交通大学;2011年
2 廖玉珍;软刻蚀方法制备细胞模板及内皮/平滑肌细胞的协同培养[D];西南交通大学;2011年
3 雷丽娟;肝素微图形的制备及对细胞行为的影响[D];西南交通大学;2011年
4 何婷婷;等离子体聚丙烯酸薄膜表面纤维连接蛋白的固定及其内皮细胞粘附行为[D];西南交通大学;2011年
5 樊珊;钛氧薄膜材料表面层粘连蛋白与纤连蛋白联合固定以及内皮化研究[D];西南交通大学;2008年
6 郑楠;几种无机材料表面微沟槽的制备及其生物相容性研究[D];西南交通大学;2009年
7 陈诚;利用自组装技术在钛表面定向固定CD34抗体及其内皮化诱导研究[D];西南交通大学;2009年
，本文编号：2266023
本文链接：https://www.wllwen.com/yixuelunwen/swyx/2266023.html
