肝素-SDF-1α聚电解质多层膜修饰大鼠脱细胞主动脉带瓣管道的组织工程研究

发布时间：2018-06-06 17:49

本文选题：基质细胞衍生因子-1α + 聚电解质多层膜　；参考：《上海交通大学》2014年博士论文

【摘要】：第一部分大鼠脱细胞主动脉带瓣管道的构建目的:瓣膜替换手术是目前对于心脏瓣膜疾病最为有效的治疗手段。但临床上所使用的人工瓣膜在生物相容性或耐久性方面存在着一定的缺陷。而组织工程瓣膜(TEHV)具有自我修复、重构以及在体内生长的优点,有可能成为传统人工瓣膜的替代者。本研究主要比较了不同脱细胞方法对于大鼠主动脉带瓣管道的脱细胞效果以及保护细胞外基质的能力,以寻求更好的办法来制备组织工程瓣膜支架。材料和方法:无菌获取大鼠主动脉带瓣管道,随机分为四组,根据脱细胞方案不同分为0.25%Triton-X100+0.25%脱氧胆酸钠(SD)组(TS组)、1%十二烷基磺酸钠组(SDS组)、0.5%Triton-X100+0.5%SD+0.5%SDS(STS组),未脱细胞的带瓣管道作为对照组。处理完成后,通过组织学HE染色评估脱细胞效果以及纤维结构的保存情况,采用DNA含量测定定量分析核酸残留情况,并确定最佳的脱细胞方案,最后通过扫描电镜评估进一步明确脱细胞效果以及纤维支架排列情况。结果:TS组以及SDS组带瓣管道细胞成分去除不完全,纤维结构破坏明显,而STS组带瓣管道脱细胞完全,纤维结构得到很好地保留。结论:联合应用Triton-X100、SD以及SDS制备的大鼠脱细胞主动脉带瓣管道细胞成分去除完全,纤维结构完整,为后续研究提供了良好的瓣膜支架材料。第二部分PEM修饰大鼠脱细胞主动脉带瓣管道及其体外相容性研究目的:脱细胞心脏瓣膜支架是构建组织工程人工瓣膜的支架材料之一。然而脱细胞心脏瓣膜支架残留的致凝性、免疫原性以及体内重构的缓慢导致其出现了早期衰败。为了克服这些缺陷以及促进再细胞化,构建具有良好生物相容性以及使用寿命的组织工程瓣膜,我们采用肝素-SDF-1α聚电解质多层膜(PEM)涂层大鼠脱细胞主动脉带瓣管道,并且体外检测其血小板和细胞相容性。材料和方法:肝素以及SDF-1α交替dip-coating在供体的U型主动脉带瓣管道表面形成PEM。通过免疫荧光检测明确肝素-SDF-1αPEM在带瓣管道表面的包被。通过血小板粘附实验以及LDH测定评估其抗血小板功能。分离、培养大鼠骨髓间充质干细胞(BMSCs),评估表面修饰对BMSCs粘附、增殖以及迁移能力的影响。结果:免疫荧光检测显示PEM包被在大鼠脱细胞带瓣管道表面。体外研究表明肝素-SDF-1αPEM显著降低了血小板在带瓣管道上的粘附从而改善了管道的血小板相容性,同时BMSCs在带管道上的粘附、增殖以及迁移能力也得到了显著增强。结论:肝素-SDF-1αPEM能够涂层在大鼠脱细胞带瓣管道表面,经过修饰的带瓣管道表现出较好的血液相容性,同时BMSCs在管道上的粘附、增殖以及迁移能力也得到了明显改善。第三部分PEM修饰大鼠脱细胞主动脉带瓣管道体内再细胞化研究目的:我们之前的研究已经证实了肝素-SDF-1αPEM修饰的大鼠脱细胞主动脉带瓣管道的生物相容性,而肝素-SDF-1αPEM修饰的脱细胞带瓣管道的再细胞化特性尚不明确。在本部分研究中,我们将修饰后的脱细胞带瓣管道移植到大鼠的肾下腹主动脉,评价其体内的功能以及再细胞化情况。材料和方法:体内研究中,我们将包被以及未包被肝素-SDF-1αPEM的脱细胞带瓣管移植到大鼠的肾下腹主动脉。12只大鼠被分为两组,PEM-DVC组(n=6):采用肝素-SDF-1αPEM修饰的脱细胞带瓣管道进行移植;Un DVC组(n=6):采用未包被的脱细胞带瓣管道进行移植。为了评估管道移植后的功能,我们在移植术后第1天以及4周时进行超声检测,同时在术后2周以及4周时进行Micro-CT检测。在带瓣管道移植后4周时将移植的带瓣管道取出,通过组织学以及免疫组织化学检测明确纤维结构的保存以及再细胞化情况。结果:不同时间点的超声以及Micro-CT检测结果显示PEM-DVC组以及Un DVC组带瓣管道移植后均保持通畅,无明显血栓、狭窄以及动脉瘤形成。移植4周后带瓣管道组织学检测显示两组带瓣管道胶原以及弹力纤维结构均完整。但是,荧光免疫组化结果表明PEM-DVC在体内能够更好的自体内皮化并能够促进内皮祖细胞在管道的募集。结论:肝素-SDF-1αPEM修饰脱细胞带瓣管道能够促进管道内膜层的再内皮化,使得管道拥有理想的血液相容性以及生理功能,为组织工程瓣膜的构建提供新的理论基础。
[Abstract]:The construction of the first part of the rat aorta with the aortic valve: valve replacement is the most effective treatment for heart valve disease. However, there are some defects in the biocompatibility or durability of the prosthetic valve used clinically, and the tissue Cheng Banmo (TEHV) is self repairing, reconstructing, and reconstructing as well. The advantages of growth in the body may be a substitute for the traditional artificial valve. This study mainly compares the decellular removal efficiency of the aortic valve conduit and the ability to protect the extracellular matrix in rat aorta, in order to find a better way to prepare the tissue engineering valve scaffold. Materials and methods: aseptic acquisition of rat main. The arterial valve tube was divided into four groups randomly. According to the different cell schemes, they were divided into 0.25%Triton-X100+0.25% deoxycholic acid sodium (SD) group (group TS), 1% sodium alkyl sulfonate group (group SDS), 0.5%Triton-X100+0.5%SD+0.5%SDS (group STS), and no acellular valve tube as the control group. After the treatment was completed, the cell removal efficiency was evaluated by histological HE staining. The preservation of fruit and fiber structure, quantitative analysis of nucleic acid residue by DNA content, and the best method of cell removal. Finally, the effect of cell removal and the arrangement of fibrous scaffold were further clarified by scanning electron microscope. Results: the removal of cell components in group TS and SDS group was incomplete and the fibrous structure was destroyed. It was found that the STS group was completely deformed and the fiber structure was well preserved. Conclusion: the combined use of Triton-X100, SD and SDS for the removal of the cell components of the rat aorta with the aortic valve was complete and the fiber structure was complete, which provided a good membrane scaffold material for the follow-up study. Second parts of the PEM modified rat were the main decellular host. Objective to study the compatibility of arterial valved conduit and in vitro Objective: the acellular heart valve scaffold is one of the scaffolding materials for the construction of tissue engineered prosthetic valves. However, the early decay of the decellular cardiac valve scaffold remains caused by the deactivation, immunogenicity, and the slow reconstruction of the body. Cytochemistry, construction of tissue engineering valve with good biocompatibility and life expectancy, we used heparin -SDF-1 alpha polyelectrolyte multilayer film (PEM) to coat the rat aortic valve ducts, and in vitro test its platelet and cell compatibility. Materials and methods: hepatin and SDF-1 alpha alternating dip-coating in the donor's U type initiative PEM. was detected by immunofluorescence to determine the inclusion of heparin -SDF-1 alpha PEM on the surface of the petal tube by immunofluorescence. The platelet adhesion test and LDH assay were used to evaluate the antiplatelet function. The rat bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured to evaluate the effect of surface repair on the adhesion, proliferation and migration of BMSCs. Results: the immunofluorescence test showed that the PEM packet was on the surface of the rat decellular valve tube. In vitro study showed that heparin -SDF-1 alpha PEM significantly reduced the adhesion of platelets on the valve tube and improved the platelets compatibility of the pipes. Meanwhile, the adhesion, proliferation and migration ability of BMSCs in the pipeline were also significantly enhanced. The heparin -SDF-1 alpha PEM can be coated on the surface of the rat decellularized petal tube, and the modified band tube shows good blood compatibility. At the same time, the adhesion, proliferation and migration of BMSCs on the pipe have also been obviously improved. Third the aim of the re cell study of the main artery petal conduit of the PEM modified rats: Our previous study has demonstrated the biocompatibility of the heparin -SDF-1 alpha PEM modified rat aortic valved conduit, while the re cell characteristics of the heparin -SDF-1 alpha PEM modified decellular valve tube are not clear. In this study, we transplanted the modified deacellular ducts to the lower abdominal initiative of rats. Pulse, evaluation of the function and re cell situation in the body. Materials and methods: in the study in vivo, we were divided into two groups,.12 rats,.12 rats were divided into two groups, group PEM-DVC (n=6): heparin -SDF-1 alpha PEM modified acellular conduit. Un DVC group (n=6): transplantation of uncovered acellular conduit. In order to evaluate the function of the tube after transplantation, we performed ultrasound examination at first and 4 weeks after the transplantation, and performed the Micro-CT test at 2 and 4 weeks after the operation. The graft conduit was removed at 4 weeks after the valve transplantation. The results of ultrasound and Micro-CT detection at different time points showed that the PEM-DVC group and the Un DVC group were all smooth after the tube transplantation, and there was no obvious thrombus, stenosis and the formation of aneurysm. 4 weeks after transplantation, the histological examination of the petal pipe showed that two The results showed that PEM-DVC could improve endothelialization in the body and promote the recruitment of endothelial progenitor cells in the pipeline. Conclusion: heparin -SDF-1 alpha PEM modification of decellular ducts can promote the re endothelialization of the tube intima and make the pipeline own The ideal blood compatibility and physiological function provide a new theoretical basis for tissue engineering valve construction.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R654.2;R318.11

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