乳液电纺论文：载质粒电纺纤维膜作为血管组织工程支架的研究.doc

乳液电纺论文：载质粒电纺纤维膜作为血管组织工程支架的研究【中文摘要】静电纺丝是利用静电力使得聚合物溶液或溶体喷射纤维的技术,近年来受到广泛的关注。静电纺丝可以制备出连续均一的纳米级纤维,具有模拟了细胞外基质中胶原纤维的纳米尺度结构和高比表面积,利于细胞的黏附和生长。因此,电纺纤维在组织工程中显示出很大的应用潜能。电纺纤维作为组织工程支架,支架的降解可促进新组织向内生长,而降解速度需要与新组织的形成速度吻合,这样才能提够一个从支架到组织的完美转换。因此,纤维支架的降解速度等需要着重考虑,使其满足具体应用的要求。本文旨在研究与聚乳酸-聚乙二醇共聚物(PELA)纤维膜在不同条件下的降解行为,结果表明纤维膜在磷酸缓冲液(PBS)、模拟矿化液(SBF)和细胞培养液中的降解行为没有明显差异。而将细胞加入培养介质中时,纤维膜的重量损失和分子量损失变大,而分子量多分散性没有明显变化。与体外无细胞参与的降解条件相比,纤维膜经皮下植入后在初始阶段的降解速度比较快,而在后期降解速度变慢。结果表明体内植入后的降解行为与在细胞种植与纤维膜上的降解行为相似,提供了一个有效的用于预测体内降解行为的体外方式。血管化是构建工程化组织中的一个重要问题,再生组织中若无法提供充足的营养供应和排出代谢废物将无法存活。本论文通过乳液电纺方式制备携载一种或两种具有血管生成因子：血管内皮生长因子(VEGF)或碱性成纤维细胞生长因子(bFGF)编码质粒的核壳纤维。体外释放实验发现核壳结构减少了突释效应,pDNA持续释放近1个月。将人脐静脉细胞(HUVEC)接种于支架上,考察了细胞的黏附和增殖、细胞的转染效率及细胞外基质分泌,结果表明纤维支架具有促进内皮细胞增殖、胶原和层粘蛋白等基质的分泌,在28时间内持续转染和表达目标蛋白。初步评价了载质粒纤维膜促进血管再生的能力,将纤维膜植入大鼠皮下,结果表明,与浸润质粒的纤维膜相比,4周时载质粒纤维膜诱导形成的血管数目增加了100%,显示了携载质粒纤维膜作为血管组织工程支架的潜力。【英文摘要】Electrospinning has become the most popular technique in recent years by the use of static electricity to draw fibers from a polymer solution or melt. Electrospinning generates uniform and continuous fibers with diameters down to a nanoscale dimension, which closely mimic the size scale of fibrous proteins found in natural extracellular matrix (ECM), and have a very high fraction of surface available to interact with cells. Therefore, electrospun fibers have gained popularity with the tissue engineering community as potential scaffolds for regeneration of cartilage, bone, skin, and blood vessels.As for the tissue engineering scaffolds, the fiber degradation is essential to enhance the ingrowth of new tissue, while the degradation rates should be matched with the rate of neo-tissue formation so as to provide a smooth transition of the load from the scaffold to the tissue. Therefore, the biodegradability should be individually tailored to meet the requirements of specific application when considering using electrospun fibers in biomedical areas. The present study aims to comprehensively investigate the effects that cells and in vivo implantation have on the degradation behaviors, compared with commonly processed degradation in buffer solutions. During the investigational time period, there was no significant difference in the degradation behaviors after incubation fibrous mats into phosphate buffer saline, simulated body fluid and cell culture media. However, significantly higher mass loss of fibrous mats, lower molecular weight reduction and less significant increase in the molecular weight polydispersity were found after inoculation of cells into degradation environment. After cell seeding on the fibrous mats, the tight attachment of cells on fibers further enhanced the degradation process. Compared with in vitro cell-free degradation medium, the subcutaneous implantation of fibrous mats led to significantly higher degradation rate at the initial stage, but slower degradation at the later stage. It was indicated that the degradation behaviors after in vivo implantation was close to those after cell culture on fibrous mats, thus providing an effective in vitro tool to predict in vivo degradation profiles of electrospun fibers, and helping researchers match them with specific purposes.One of the most important issues for engineered tissues is the vascularization of the constructs, since a tissue that is more than a few millimeters in size generally cannot survive by only the diffusion of nutrients and metabolic products. Hence, acceleration of the angiogenesis rate is urgently required.Core-shell structured fibers encapsulated pDNA encoding vascular-endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) or both of them were prepared by emulsion electrospining. In vitro release study indicated that the core-shell structure inhibited the burst elease and prvide a sustained release of pDNA for around 1 month. Human umbilical vein endothelial cells (HUVEC) were seeded on pDNA-loaded firbousscaffolds, indicating the capabilities of promoting endothelial cell proliferation and ECM secretion by the autocrine of growth factors, compared with pDNA-infiltrared fibrous mats. An effective cell transfection and target protein expression proceeded for over 28 d. The pDNA-loaded fibrous scaffolds were subcutaneously implanted into rats to study their angiogenesis via macroscopic observation, hematoxylin-eosin staining and immunohistochemical staining. The results demonstrated that the incorporation of pDNA polyplexes could effectively enhance the angiogenesis of the implanted fibrous mats, demonstrating the potential application as scaffolds in vascular tissue engineering.【关键词】乳液电纺 核壳结构 降解行为 血管组织工程 基因释放【英文关键词】degradation behaviors emulsion electrospinning core-shell structure vascular tissue engineering gene delivery【目录】载质粒电纺纤维膜作为血管组织工程支架的研究摘要7-8Abstract8-9目录10-13第1章 绪论13-251.1 静电纺丝技术13-161.1.1 静电纺纤维的特点131.1.2 核壳纤维的特点及制备方法13-151.1.3 乳液电纺制备核壳纤维的原理及研究现状15-161.2 静电纺纤维组织工程支架16-181.2.1 静电纺纤维组织工程支架的特点及优势161.2.2 静电纺纤维组织工程支架的研究现状16-171.2.3 载活性物质静电纺纤维组织工程支架的研究进展17-181.3 载基因组织工程支架18-201.3.1 载基因的优势18-191.3.2 载基因组织工程支架的研究进展19-201.3.3 静电纺纤维载基因的研究进展201.4 血管组织工程20-231.4.1 血管组织工程的进展21-221.4.2 静电纺纤维作为血管组织工程支架的研究进展22-231.5 课题的立题意义和研究内容23-251.5.1 本课题的来源231.5.2 本课题的研究目的和意义231.5.3 本课题的研究内容23-241.5.4 本课题的创新点24-25第2章 电纺纤维支架的体内外的降解行为25-372.1 实验部分25-272.1.1 实验材料252.1.2 电纺PELA纤维25-262.1.3 PELA电纺纤维的表征262.1.4 PELA电纺纤维的灭菌及细胞培养262.1.5 PELA电纺纤维的体外降解行为26-272.1.6 PELA电纺纤维的体内降解行为272.1.7 PELA电纺纤维降解行为的表征272.1.8 统计学分析272.2 实验结果27-362.2.1 电纺纤维PELA的表征27-282.2.2 灭菌方式对电纺纤维PELA的影响28-292.2.3 电纺纤维PELA在体外降解后的形貌变化29-312.2.4 电纺纤维PELA在体外的降解行为31-332.2.5 电纺纤维PELA在体内的降解行为33-362.3 本章小结36-37第3章 携载PDNA-PEI复合粒子电纺纤维的制备及表征37-483.1 实验部分37-423.1.1 实验材料373.1.2 质粒PVEGF和PBFGF的提取及纯化37-383.1.3 质粒DNA的定量及定性分析38-393.1.4 PDNA-PEI复合粒子的制备393.1.5 PDNA-PEI复合粒子的表征39-403.1.6 载PDNA-PEI复合粒子电纺纤维的制备40-413.1.7 载PDNA-PEI复合粒子电纺纤维的表征413.1.8 电纺纤维中质粒DNA的表征41-423.1.9 电纺纤维中质粒DNA的体外释放行为423.1.10 统计学分析423.2 结果与讨论42-473.2.1 纯化PVEGF的表征结果42-433.2.2 PDNA-PEI复合粒子的表征43-443.2.3 载PDNA-PEI复合粒子电纺纤维的形貌与结构44-453.2.4 电纺纤维中质粒DNA的表征45-463.2.5 电纺纤维中质粒DNA的体外释放行为46-473.3 本章小结47-48第4章 携载PDNA-PEI复合粒子电纺纤维的生物学评价48-654.1 实验部分48-534.1.1 实验材料48-494.1.2 细胞在纤维膜上的接种494.1.3 细胞在纤维膜的生长行为49-504.1.4 纤维中包裹质粒PDNA对细胞的转染504.1.5 纤维膜上细胞的VEGF表达量50-514.1.6 纤维膜上细胞的基质分泌51-524.1.7 体内植入实验524.1.8 组织学观察52-534.1.9 统计学分析534.2 实验结果53-644.2.1 细胞在纤维膜上的生长行为53-554.2.2 纤维膜上细胞的VEGF表达量55-564.2.3 纤维膜上细胞的转染效率56-584.2.4 纤维膜上细胞的基质分泌58-614.2.5 细胞与支架间的相互作用61-634.2.6 成熟血管的密度63-644.3 本章小结64-65全文结论65-66致谢66-67参考文献67-74攻读硕士学位期间发表的论文74-75