层层组装构建细胞选择性界面的研究
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摘要
聚对苯二甲酸乙二醇酯(PET)是广泛应用于大、中型动脉替代物的人工血管材料,但当其应用于小口径(直径小于6 mm)的人工血管时,常由于血栓的形成和平滑肌细胞过度增生而导致移植的失败。内皮化是解决这一问题的重要途径。本论文以层层静电自组装技术为手段,以PET片作基底模型,构建了具有溶胀结构的阻抗非特异性细胞粘附的多层膜体系,并在多层膜表面分别固定了能特异性促进内皮细胞粘附的生物活性抗体分子和功能性短肽片段,研究了多层膜表面内皮细胞和平滑肌细胞的粘附行为。
本文首先以模拟细胞膜表面“糖质衣”阻抗非特异性细胞粘附的功能为出发点,采用肝素(HEP)和壳聚糖(CHI)在PET表面层层组装,设计具有阻抗非特异性细胞粘附功能多层膜界面。石英晶体微天平(QCM-D)测试表明肝素、壳聚糖成功的交替组装在晶片表面。体外抗凝血实验显示肝素的协同作用导致本来致凝血的壳聚糖层表现出良好的抗凝血性能。对多层膜表面内皮细胞和平滑肌细胞培养的研究显示,两种细胞的粘附率和增殖率均显著低于TCPS对照,细胞形态呈圆球形未铺展状态,细胞面积比TCPS上细胞面积小,表明多层膜表面能有效阻抗内皮细胞和平滑肌细胞粘附。随多层膜层数增多,阻抗细胞粘附特性(即细胞惰性)略微增强,说明多层膜的溶胀结构导致其阻抗内皮细胞和平滑肌细胞的粘附,结构越溶胀,细胞惰性越强,是一种良好的阻抗非特异性细胞粘附的界面。
其次本文以细胞表面抗原特异性结合抗体分子的生物机制为启示,在具有阻抗非特异性细胞粘附的(肝素/壳聚糖)多层膜表面固定CD34抗体,利用CD34抗体与内皮祖细胞表面抗原的特异性相互作用达到促进内皮细胞选择性粘附的目的。采用静电吸附的方式,在带正电的壳聚糖作最外层的多层膜表面固定带负电的CD34抗体。QCM-D测试表明抗体固定密度约为4 ng╱cm~2。QCM-D稳定性测试表明,固定了抗体的多层膜在模拟人体血液流速环境下的PBS溶液中冲洗2小时后,仍然保持稳定。对抗体功能化的多层膜表面内皮细胞培养的研究显示,固定了抗体的多层膜可以提高内皮细胞的粘附率。平滑肌细胞体外培养结果表明,抗体功能化的多层膜仍能有效阻抗平滑肌细胞的粘附,说明CD34抗体分子只对内皮细胞有特异性相互作用,对平滑肌细胞没有相互作用。该研究构建了可促进内皮细胞粘附,阻抗平滑肌细胞粘附的具有内皮细胞选择性的界面。
最后本文以细胞表面的整合素特异性识别短序列多肽片段的生物原理为启示,在具有阻抗非特异性细胞粘附的多层膜表面固定REDV功能性短肽片段,利用REDV与内皮细胞表面整合素的特异性识别作用达到促进内皮细胞选择性粘附的目的。采用层层静电自组装的方式将共价接枝了REDV的壳聚糖负载到(肝素/壳聚糖)多层膜表面,并用茚三酮-紫外光谱分析、石英晶体微天平测试证明了多层膜表面成功固定了REDV短肽。对REDV功能化的多层膜表面内皮细胞和平滑肌细胞培养的研究显示,固定了REDV短肽的多层膜可以促进内皮细胞的粘附,但仍阻抗平滑肌细胞的粘附。对改变氨基酸序列的另一种短肽:REVD功能化的多层膜表面内皮细胞培养结果显示,REVD功能化多层膜对内皮细胞起阻抗粘附作用。说明多层膜表面促进内皮细胞的粘附是由于固定了REDV功能性短肽片段导致的,REDV只对内皮细胞具有特异性识别作用,对平滑肌细胞没有识别功能。该研究构建了另一种可促进内皮细胞粘附,阻抗平滑肌细胞粘附的具有内皮细胞选择性的界面。
Poly-(ethylene terephthalate) (PET) has been used successfully in treating the pathology of large diameter arteries (>6 mm, inner diameter), but has not been proven to be successful in replacing small diameter blood vessels (<6 mm). Incomplete coverage of endothelial cells (ECs) on the vascular graft surfaces, the smooth muscle cells (SMCs) hyperplasia and the subsequent thrombus generation are the main reasons for the long term failure of small-diameter vascular grafts. Endothelialization is one of the approaches to solve this problem. In this thesis, surface modification of PET was carried out via layer-by-layer self-assembly technique. A multilayered film system with low modulus and swollen structure was constructed, which could hinder the non-specific cell adhesion. A bioactive antibody and a functional peptide were respectively immobilized on the multilayered film surface, which could promote the specific adhesion of ECs. The short-term adhesion of ECs and SMCs were studied on multilayered film surfaces.
Firstly, heparin (HEP) and chitosan (CHI) were assembled to construct multilayered films on PET surfaces. This multilayered film system with swollen structures was constructed to obstruct the non-specific cell adhesion. Layer-by-layer assembly process of heparin and chitosan was monitored by Quartz Crystal Microbalance-Dissipation (QCM-D). In vitro anticoagulant test verified that the initial coagulated chitosan became anticoagulated under the collaboration effect with heparin, which was a strong anticoagulant. The culture results of ECs and SMCs on multilayered film surfaces suggested that both the cells had poor adhesion and proliferation behaviors, compared to TCPS. Both the cells showed round un-proliferated images and the cell areas were both smaller than the ones on TCPS, which revealed that the multilayered films could obstruct the adhesion of ECs and SMCs. The films became slightly more cytophobic (or inert) when the number of assemble layers increased, which might caused by the swollen structure of multilayered films. More swollen the structure was, more inert the films became. The (HEP/CHI) multilayered films have good potential in obstructing non-specific cell adhesion.
Secondly, CD34 antibody was immobilized on the surface of (HEP/CHI) multilayered films, which could promote the specific adhesion of ECs by utilizing the specific interaction between antibodies and endothelial progenitor cell (EPC) antigens. By electrostatic interaction, the negative CD34 antibody was immobilized on the surface of positive chitosan-terminated multilayered films. The desity of immobilized antibodies was about 4 ng/cm~2 by QCM-D test. The stability test indicated that the CD34 antibody-modified multilayered films could remain stable after 2 hours by PBS flushing, which simulated the human blood flow environment. The ECs' culture results of CD34 antibody-functional multilayered films revealed that the films could increase the adhesion rate of ECs. SMCs were prevented to adhere on CD34 antibody-modified films by cell cultured test, which indicated CD34 antibodies could specifically interact with ECs rather than SMCs. The (HEP/CHI) multilayered film immobilized with CD34 antibodies could promote the ECs to adhere and hinder the SMCs to adhere, which is an EC selective interface.
Finally, REDV functional peptide was immobilized on the surface of (HEP/CHI) multilayered films, which could promote the specific adhesion of ECs by utilizing the specific recognition between peptides and EC surface integrins. The REDV-covalent chitosan solution was assembled on (HEP/CHI) multilayered film surfaces via layer-by-layer self-assembly technique. The immobilization process of REDV peptide was monitored by ninhydrin-UV spectra and QCM-D test. The ECs and SMCs' culture results on REDV-functional multilayered films revealed that the films could improve the adhesion rate of ECs and obstruct SMCs to adhere. The cell adhesion behaviors of ECs cultured on another peptide-REVD suggested that the REVD had no promotion of EC adhesion. The results revealed that (HEP/CHI) multilayered films could improve the adhesion of ECs because of the immobilization of REDV. REDV peptides could specifically interact with ECs rather than SMCs. The multilayered film immobilized with REDV peptides is another EC selective interface.
本文首先以模拟细胞膜表面“糖质衣”阻抗非特异性细胞粘附的功能为出发点,采用肝素(HEP)和壳聚糖(CHI)在PET表面层层组装,设计具有阻抗非特异性细胞粘附功能多层膜界面。石英晶体微天平(QCM-D)测试表明肝素、壳聚糖成功的交替组装在晶片表面。体外抗凝血实验显示肝素的协同作用导致本来致凝血的壳聚糖层表现出良好的抗凝血性能。对多层膜表面内皮细胞和平滑肌细胞培养的研究显示,两种细胞的粘附率和增殖率均显著低于TCPS对照,细胞形态呈圆球形未铺展状态,细胞面积比TCPS上细胞面积小,表明多层膜表面能有效阻抗内皮细胞和平滑肌细胞粘附。随多层膜层数增多,阻抗细胞粘附特性(即细胞惰性)略微增强,说明多层膜的溶胀结构导致其阻抗内皮细胞和平滑肌细胞的粘附,结构越溶胀,细胞惰性越强,是一种良好的阻抗非特异性细胞粘附的界面。
其次本文以细胞表面抗原特异性结合抗体分子的生物机制为启示,在具有阻抗非特异性细胞粘附的(肝素/壳聚糖)多层膜表面固定CD34抗体,利用CD34抗体与内皮祖细胞表面抗原的特异性相互作用达到促进内皮细胞选择性粘附的目的。采用静电吸附的方式,在带正电的壳聚糖作最外层的多层膜表面固定带负电的CD34抗体。QCM-D测试表明抗体固定密度约为4 ng╱cm~2。QCM-D稳定性测试表明,固定了抗体的多层膜在模拟人体血液流速环境下的PBS溶液中冲洗2小时后,仍然保持稳定。对抗体功能化的多层膜表面内皮细胞培养的研究显示,固定了抗体的多层膜可以提高内皮细胞的粘附率。平滑肌细胞体外培养结果表明,抗体功能化的多层膜仍能有效阻抗平滑肌细胞的粘附,说明CD34抗体分子只对内皮细胞有特异性相互作用,对平滑肌细胞没有相互作用。该研究构建了可促进内皮细胞粘附,阻抗平滑肌细胞粘附的具有内皮细胞选择性的界面。
最后本文以细胞表面的整合素特异性识别短序列多肽片段的生物原理为启示,在具有阻抗非特异性细胞粘附的多层膜表面固定REDV功能性短肽片段,利用REDV与内皮细胞表面整合素的特异性识别作用达到促进内皮细胞选择性粘附的目的。采用层层静电自组装的方式将共价接枝了REDV的壳聚糖负载到(肝素/壳聚糖)多层膜表面,并用茚三酮-紫外光谱分析、石英晶体微天平测试证明了多层膜表面成功固定了REDV短肽。对REDV功能化的多层膜表面内皮细胞和平滑肌细胞培养的研究显示,固定了REDV短肽的多层膜可以促进内皮细胞的粘附,但仍阻抗平滑肌细胞的粘附。对改变氨基酸序列的另一种短肽:REVD功能化的多层膜表面内皮细胞培养结果显示,REVD功能化多层膜对内皮细胞起阻抗粘附作用。说明多层膜表面促进内皮细胞的粘附是由于固定了REDV功能性短肽片段导致的,REDV只对内皮细胞具有特异性识别作用,对平滑肌细胞没有识别功能。该研究构建了另一种可促进内皮细胞粘附,阻抗平滑肌细胞粘附的具有内皮细胞选择性的界面。
Poly-(ethylene terephthalate) (PET) has been used successfully in treating the pathology of large diameter arteries (>6 mm, inner diameter), but has not been proven to be successful in replacing small diameter blood vessels (<6 mm). Incomplete coverage of endothelial cells (ECs) on the vascular graft surfaces, the smooth muscle cells (SMCs) hyperplasia and the subsequent thrombus generation are the main reasons for the long term failure of small-diameter vascular grafts. Endothelialization is one of the approaches to solve this problem. In this thesis, surface modification of PET was carried out via layer-by-layer self-assembly technique. A multilayered film system with low modulus and swollen structure was constructed, which could hinder the non-specific cell adhesion. A bioactive antibody and a functional peptide were respectively immobilized on the multilayered film surface, which could promote the specific adhesion of ECs. The short-term adhesion of ECs and SMCs were studied on multilayered film surfaces.
Firstly, heparin (HEP) and chitosan (CHI) were assembled to construct multilayered films on PET surfaces. This multilayered film system with swollen structures was constructed to obstruct the non-specific cell adhesion. Layer-by-layer assembly process of heparin and chitosan was monitored by Quartz Crystal Microbalance-Dissipation (QCM-D). In vitro anticoagulant test verified that the initial coagulated chitosan became anticoagulated under the collaboration effect with heparin, which was a strong anticoagulant. The culture results of ECs and SMCs on multilayered film surfaces suggested that both the cells had poor adhesion and proliferation behaviors, compared to TCPS. Both the cells showed round un-proliferated images and the cell areas were both smaller than the ones on TCPS, which revealed that the multilayered films could obstruct the adhesion of ECs and SMCs. The films became slightly more cytophobic (or inert) when the number of assemble layers increased, which might caused by the swollen structure of multilayered films. More swollen the structure was, more inert the films became. The (HEP/CHI) multilayered films have good potential in obstructing non-specific cell adhesion.
Secondly, CD34 antibody was immobilized on the surface of (HEP/CHI) multilayered films, which could promote the specific adhesion of ECs by utilizing the specific interaction between antibodies and endothelial progenitor cell (EPC) antigens. By electrostatic interaction, the negative CD34 antibody was immobilized on the surface of positive chitosan-terminated multilayered films. The desity of immobilized antibodies was about 4 ng/cm~2 by QCM-D test. The stability test indicated that the CD34 antibody-modified multilayered films could remain stable after 2 hours by PBS flushing, which simulated the human blood flow environment. The ECs' culture results of CD34 antibody-functional multilayered films revealed that the films could increase the adhesion rate of ECs. SMCs were prevented to adhere on CD34 antibody-modified films by cell cultured test, which indicated CD34 antibodies could specifically interact with ECs rather than SMCs. The (HEP/CHI) multilayered film immobilized with CD34 antibodies could promote the ECs to adhere and hinder the SMCs to adhere, which is an EC selective interface.
Finally, REDV functional peptide was immobilized on the surface of (HEP/CHI) multilayered films, which could promote the specific adhesion of ECs by utilizing the specific recognition between peptides and EC surface integrins. The REDV-covalent chitosan solution was assembled on (HEP/CHI) multilayered film surfaces via layer-by-layer self-assembly technique. The immobilization process of REDV peptide was monitored by ninhydrin-UV spectra and QCM-D test. The ECs and SMCs' culture results on REDV-functional multilayered films revealed that the films could improve the adhesion rate of ECs and obstruct SMCs to adhere. The cell adhesion behaviors of ECs cultured on another peptide-REVD suggested that the REVD had no promotion of EC adhesion. The results revealed that (HEP/CHI) multilayered films could improve the adhesion of ECs because of the immobilization of REDV. REDV peptides could specifically interact with ECs rather than SMCs. The multilayered film immobilized with REDV peptides is another EC selective interface.
引文
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