以脱细胞脐动脉为支架构建组织工程血管的研究

发布时间：2018-04-02 23:35

本文选题：毛囊　切入点：间充质干细胞　出处：《吉林大学》2014年博士论文

【摘要】：组织工程血管的构建是组织工程研究领域的重点和难点，其成功构建不仅为可供移植的材料提供了新的来源，更为临床上诸多心血管疾病的患者带来治愈的希望。本研究拟以人毛囊间充质干细胞为种子细胞，以脱细胞胎儿脐动脉为支架材料，，体外构建组织工程血管，所得结果如下：（一）应用拔取成人毛发的方法，从中分离获得人毛囊间充质干细胞，通过显微镜观察、流式细胞分析的方法，从形态学、分子标记物方面检测一般特性；通过诱导成骨、成脂肪及成软骨检测其多向分化潜能的生物学行为；通过流式细胞分析、免疫荧光染色及实时定量PCR对人毛囊间充质干细胞来源的平滑肌细胞进行鉴定，发现其表达成熟平滑肌细胞的特异性标志物。从而证明人毛囊间充质干细胞可以作为构建组织工程血管的种子细胞。（二）获取在临床上废弃的胎儿脐动脉，经胰蛋白酶和十二烷基硫酸钠联合处理去掉脐动脉的细胞成分作为组织工程血管的支架。组织学观察发现该动脉细胞成分已经完全去除，但胶原和弹力纤维的结构仍保持完好。扫描电镜进一步确定纤维互相交织，为以后种子细胞的铺展、迁徙、分化提供合适的生存环境。细胞化学及生物化学分析还发现脱细胞脐动脉细胞外基质成分依然存在，胶原蛋白及弹力纤维的含量并未发生明显变化。将脱细胞前后的脐动脉进行生物力学的比较发现脱细胞的脐动脉具有较好的生物力学性质。可见利用临床废弃组织，经脱细胞处理后作为组织工程血管的支架，具有很好的应用前景。（三）将人毛囊间充质干细胞接种在脱细胞脐动脉支架上构建组织工程血管，组织学确定种子细胞黏附并迁移至血管的中膜，免疫组织化学染色检测平滑肌标志物α-actin和calponin呈阳性表达。重要的是构建的组织工程血管已经表现出对血管活性物质如氯化钾、去甲肾上腺素及硝普钠的反应活性，从而成功构建了在组织结构和生理功能与天然血管极为接近的组织工程血管。综上所述，我们建立了高效的分离、纯化、培养及扩增人毛囊间充质干细胞的技术体系；制备了具有合理的空间结构及组织相容性的脱细胞动脉支架；成功构建了具有生物活性的组织工程血管，为开展无免疫排斥的个性化血管移植治疗打下坚实的实验基础。
[Abstract]:The construction of tissue engineering blood vessels is a key and difficult point in the field of tissue engineering. The successful construction of tissue engineering vessels not only provides a new source for transplantable materials, but also brings the hope of cure for many patients with cardiovascular diseases.In this study, human hair follicle mesenchymal stem cells were used as seed cells and acellular fetal umbilical artery as scaffold material to construct tissue engineered blood vessels in vitro. The results were as follows:(1) Human hair follicle mesenchymal stem cells (HMSCs) were isolated from adult hair by the method of extraction of adult hair. The general characteristics of human hair follicle mesenchymal stem cells were detected from morphology and molecular markers through microscope observation and flow cytometry.The biological behaviors of adipogenic and chondrogenic multidirectional differentiation potential were detected, and the smooth muscle cells derived from human hair follicle mesenchymal stem cells were identified by flow cytometry, immunofluorescence staining and real-time quantitative PCR.A specific marker for the expression of mature smooth muscle cells was found.It is shown that human hair follicle mesenchymal stem cells can be used as seed cells to construct tissue engineered blood vessels.(2) the fetal umbilical artery abandoned in clinic was obtained and removed from the umbilical artery by trypsin and sodium dodecyl sulfate as stents for tissue engineering vessels.Histological observation showed that the cellular components of the artery had been completely removed, but the structure of collagen and elastic fibers remained intact.Scanning electron microscopy (SEM) further confirmed that the fibers intertwined with each other, providing a suitable living environment for seed cells to spread, migrate and differentiate in the future.Cytochemistry and biochemistry analysis also showed that extracellular matrix of acellular umbilical artery still existed, and the contents of collagen and elastic fiber did not change obviously.The biomechanical comparison of the umbilical artery before and after acellular showed that the acellular umbilical artery had better biomechanical properties.It can be seen that the scaffold of tissue engineering vessels using clinical discarded tissue and acellular treatment has a good application prospect.(3) Human hair follicle mesenchymal stem cells were inoculated on the scaffold of acellular umbilical artery to construct tissue engineered blood vessels.The expression of 伪 -actin and calponin was detected by immunohistochemical staining.It is important that the constructed tissue engineered blood vessels have shown reactive activity to vasoactive substances such as potassium chloride, norepinephrine and sodium nitroprusside.Thus, tissue engineered blood vessels which are very close to natural blood vessels in tissue structure and physiological function were successfully constructed.To sum up, we established a highly efficient technique system for isolation, purification, culture and amplification of human hair follicle mesenchymal stem cells, and prepared acellular artery stents with reasonable spatial structure and histocompatibility.A bioactive tissue engineering blood vessel was successfully constructed, which laid a solid experimental foundation for individualized vascular transplantation without immune rejection.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R318.08

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