促血管生成功能化自组装多肽的筛选及细胞学评价

发布时间：2018-04-14 21:31

本文选题：自组装多肽 + 血管生成　；参考：《清华大学》2012年硕士论文

【摘要】：功能化自组装多肽纳米纤维支架材料因其独特的设计以及良好的生物相容性和可降解性已成为了新兴的组织工程支架材料。三维支架材料里微血管系统的长入和建立是组织工程移植材料长期存活的重要保证，也是一直以来组织工程产品临床应用中所面临的重要瓶颈之一。本文设计和制备了一系列具有血管生成活性的自组装多肽纳米纤维水凝胶支架材料。从材料学角度分析自组装多肽的显微结构，从细胞学角度筛选出能够促进血管生成的多肽水凝胶。为了进一步验证多肽具有促血管生成作用，尝试了内皮细胞和平滑肌细胞的二维共培养，观察细胞在多肽水凝胶上的生长情况。从促进内皮细胞贴附增殖以及抑制其凋亡的蛋白质及生长因子中，设计了六种氨基酸序列，通过多肽固相合成法将其接枝在自组装多肽RADA16-I的末端。将1%功能化多肽与RADA16-I按1:1的体积比混合形成功能化自组装多肽。通过圆二色谱分析其二级结构，证实形成了β折叠结构；在原子力显微镜（AFM）下可以看到均匀的纳米纤维结构；在扫描电子显微镜（SEM）下，可以看到其多孔状结构与天然的细胞外基质结构十分相似，是十分理想的三维组织工程支架材料。将人脐静脉内皮细胞（HUVEC）种植在功能化自组装多肽水凝胶上，通过观察细胞的贴附、增殖、迁移及形貌分化，筛选出两种促进内皮细胞增殖和形貌分化的多肽片段——KLT（KLTWQELYQLKYKGI）和PRG（GPRGDSGYRGDS），具有促血管生成的潜能。为了进一步证实这两种多肽的促血管生成作用，尝试了内皮细胞和平滑肌细胞在二维表面上的共培养，，可以看到这两种多肽具有明显的促细胞增殖和形貌分化作用。
[Abstract]:Functional self-assembled polypeptide nanofiber scaffolds have become new scaffolds for tissue engineering because of their unique design, good biocompatibility and biodegradability.The growth and establishment of microvascular system in three-dimensional scaffolds is an important guarantee for the long-term survival of tissue engineering transplantation materials, and it is also one of the important bottlenecks in the clinical application of tissue engineering products.A series of self-assembled polypeptide nanofiber hydrogel scaffolds with angiogenic activity were designed and prepared.The microstructures of self-assembled polypeptides were analyzed from the point of view of materials, and the polypeptide hydrogels which could promote angiogenesis were screened from the perspective of cytology.In order to further verify the effect of polypeptide on angiogenesis, two dimensional co-culture of endothelial cells and smooth muscle cells was attempted to observe the growth of the cells on polypeptide hydrogel.Six amino acid sequences were designed from the proteins and growth factors that promoted the proliferation of endothelial cells and inhibited their apoptosis. They were grafted onto the end of self-assembled polypeptide RADA16-I by solid phase peptide synthesis.1% functionalized polypeptide was mixed with RADA16-I at 1:1 volume ratio to form functionalized self-assembled polypeptide.The secondary structure was analyzed by circular dichroism, and it was confirmed that the 尾 -fold structure was formed; the uniform nanofiber structure could be seen under atomic force microscope (AFM); and under scanning electron microscope (SEM),It can be seen that the porous structure is very similar to the natural extracellular matrix and is an ideal three-dimensional scaffold for tissue engineering.Human umbilical vein endothelial cells (HUVECs) were implanted on functionalized self-assembled polypeptide hydrogels. The adhesion, proliferation, migration and morphological differentiation of the cells were observed.Two kinds of polypeptide fragments, KLTWELYQLYKGI and PRGN GPRGDSGYR GDSs, were screened to promote the proliferation and morphological differentiation of endothelial cells, which have the potential of promoting angiogenesis.In order to further confirm the angiogenic effect of these two peptides, the co-culture of endothelial cells and smooth muscle cells on the two-dimensional surface was attempted. It can be seen that the two peptides have obvious effects on promoting cell proliferation and morphologic differentiation.
【学位授予单位】：清华大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08

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