【作者】 肖荣冬；

【导师】 翁国星；

【作者基本信息】 福建医科大学 ， 外科学， 2006， 博士

【摘要】 人们长期以来一直在寻求一种血管的最佳替代物,尝试了自体动静脉、人工材料、生物材料等多种方法,但这些方法均存在一些不足。组织工程概念的提出及技术的逐步成熟使在体外培植具有生物活性,结构、功能与自体血管相类似的人工血管成为可能。它标志着医学步入制造组织和器官的新时代,是生命科学发展史上的新的里程碑。本实验培养血管内皮细胞和平滑肌细胞为种子细胞,制备了天然生物复合材料,对几种生物材料的生物性能及细胞相容性进行了研究,应用组织工程的方法,制造出组织工程补片并进行了在体实验研究,同时,应用模具直接成形,冷冻干燥等技术制备了管状生物支架并应用动态培养系统对细胞在管状支架上的种植方法进行了探索。第一部分兔主动脉内皮细胞和平滑肌细胞的分离、培养目的:为构建组织工程血管提供种子细胞。方法:兔麻醉后取下主动脉,分别采用酶消化法和组织块贴壁法培养血管内皮细胞和平滑肌细胞并传代,倒置显微镜、扫描电镜、透射电镜观察和免疫组化鉴定。结果:成功的培养出血管内皮细胞和平滑肌细胞并传代。倒置显微镜下血管内皮细胞呈“鹅卵石”形态,平滑肌细胞典型的“峰-谷”状,透射电镜内皮细胞特征性W-P小体,平滑肌细胞大量肌丝,免疫组化内皮细胞Ⅷ因子(+),平滑肌细胞肌动蛋白抗体(+)。结论:所培养的血管内皮细胞和平滑肌细胞为组织工程血管的研究提供了细胞来源。第二部分组织工程支架材料的制备与生物学性能检测目的:制备复合天然生物支架材料胶原/透明质酸膜、明胶/壳聚糖膜,并评估胶原/透明质酸膜、明胶/壳聚糖膜和明胶海绵(由Pfizer corporation USA提供)这三种材料的生物学性能及组织相容性。方法:1.应用材料复合交联的实验方法构建胶原/透明质酸膜、明胶/壳聚糖膜,光镜,扫描电镜观察胶原/透明质酸膜、明胶/壳聚糖膜、明胶海绵。2.应用万能测试机测定胶原/透明质酸膜、明胶/壳聚糖膜、明胶海绵的力学性能,包括韧性指标和抗张强度,测定材料吸水率和接触角。3.在体外应用胶原酶,溶菌酶和去离子水测定体外降解特性,将支架材料种植于兔皮下,进行皮下包埋实验,分别在2周、4周、6周、8周和12周光学显微镜观察,评价材料在体内的降解情况和组织相容性。结果:1.成功制备了胶原/透明质酸膜和明胶/壳聚糖膜,光镜,电镜下观察三种材料均具有网孔状结构,胶原/透明质酸膜和明胶/壳聚糖膜孔径均匀,明胶海绵更多还原

【Abstract】 A best substitute of blood vessel has been pursued for a long time and many materials have been tried such as autologous artery or vein, artificial material , biomaterial, but no ideal substitute has been found up till now. Tissue engineering is a new and rapidly expanding field, in which a series of techniques are being developed for culturing and reconstructing a variety of tissues both in vitro and in vivo . With these techniques ,it is possible to reconstruct vascular model in vitro whose structure and function are similar to autogenous vessels.In our experiment vascular endothelial cells and smooth muscle cells were cultured for the source of seeding cells. A number of natural biological composite materials were manufactured and its biological peformance and cell compatibility were investigated. Tissue engineering patch were used for experimental research in vivo. Tubular biological vascular scaffold was made under vaccum freeze condition in a special annular model and the application of dynamic implantation in tubular scaffold was explored .Part one culture of endothelial cell and smooth muscle cell of rabbit aortaObjective: To provide seeding cells for vascular tissue engineering research. Methods: Aortas of rabbits were excised under anesthesia, vascular endothelial cells and smooth muscle cells were cultured respectively by enzyme digestion and tissue explant method, subcultured and identified by morphological characteristics under phase-contrast microscope, scanning electron microscope(SEM), transmission elctron microscope(TEM) and immunohistochemical staining. Results: Both of two kinds of cells are cultured and subcultured successfully. Vascular endothelial cells show slabstone appearance enchased arrange and smooth muscle cells“hill and vale”appearance under phase-contrast microscope. The growth appearance were showed under SEM. Vascular endothelial cells show“W-P”and smooth muscle cells“myofilament”under TEM. Immunohistochemical staining results show that endothelial cells areⅧfactor positive and smooth muscle cellsα-actin positive. Conclusion: Vascular endothelial cells and smooth muscle cells we cultured provide cell source for the research of vascuilar tissue engineering.更多还原