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摘要近年来人们致力于各种皮肤替代物的研究，为皮肤全层缺损的修复尤其是大面积深度烧伤病人的治疗带来了希望。胶原蛋白由于其良好的生物学特性已成为组织工程常用的载体材料。本研究所用的重组人源胶原蛋白(RHC)是通过基因工程法设计胶原蛋白基因序列，而后通过毕赤酵母表达系统表达生产的具有人胶原蛋白特征的蛋白质，与从动物体提取的胶原蛋白相比，具有亲水性、无病毒隐患的优点。本课题中，我们采用天然交联剂—原花青素（PA)交联的RHC-壳聚糖生物海绵，进行生物相容性研究。通过吖啶橙/溴化乙锭（AO／EB）染色，实时定量PCR（qRT-PCR），探讨了生物海绵对人脐静脉内皮细胞(HUVEC)的增殖、迁移、分化的影响。结果发现生物海绵无细胞毒性，基本不影响细胞的生长活性，侧面反映出生物海绵具有良好的细胞相容性，有望应用于皮肤组织工程。28957
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关键词 皮肤组织工程 重组人源胶原蛋白 原花青素 人脐静脉内皮细胞(HUVEC)
毕业论文设计说明书外文摘要
Title Research on the key technique of three dimensional tissue scaffolds fabrication
Abstract
In recent years, People committed to the research of varieties of skin substitutes, for the repair of full-thickness skin defect especially in the treatment of patients suffering from large deep burn. Collagen with good biological characteristics has become a common carrier material for tissue engineering.The recombinant human collagen (RHC) used in this paper is a protein with character of human collagen produced by collagen gene sequence design via gene engineering, followed by expression of Picha Pastoris system. Compared with collagen extracted from animal, it possesses the advantages of hydrophilic without virus risk. In this paper, we used a natural crosslinking agent - proanthocyanidins (PA) cross-linked RHC-chitosan scaffold to investigated its biocompatibility. Through the AO / EB staining and qRT-PCR, we explored the effects of scaffold on proliferation, migration and differentiation of Human Umbilical Vein Endothelial Cells (HUVEC).As a result, the scaffold exhibited non cytotoxicity that it did not affect the cell growth and reflected good cell compatibility. These characterization indicated that this scaffold can be considered as an ideal candidate for skin tissue engineering.
Keywords Skin tissue engineering Recombinant human collagen Proanthocyanidins Human umbilical vein endothelial cells
目 次
1 绪论 1
1.1 胶原蛋白 1
1.1.1 胶原蛋白简介 1
1.1.2 胶原蛋白结构与类型 1
1.1.3 重组人源胶原蛋白简介 2
1.1.4 重组人源胶原蛋白功能与应用 3
1.2 皮肤组织工程 3
1.2.1 组织工程简介 3
1.2.2 组织工程中细胞培养 3
1.2.3 皮肤组织工程发展 4
1.2.4 重组人源胶原蛋白在皮肤组织工程中的应用 5
1.3 胶原三文多孔支架常用交联方法 5
1.3.1 常用交联方法 6
1.3.2 天然交联剂 6
1.4 课题主要研究内容与意义 8
2 重组人源胶原蛋白-壳聚糖生物海绵对HUVEC细胞的影响 9
2.1 材料与方法 9
2.1.1 实验材料与试剂 9
2.1.2 主要仪器及设备 10
2.1.3 细胞二文静态培养 11
2.1.4 细胞三文静态培养 11