RGD肽固定对组织工程心脏瓣膜构建的作用

Effects of RGD Peptides on Constructing Tissue Engineering Heart Valves in Vitro

目的探讨促黏附分子 RGD 肽(精氨酸-甘氨酸-天冬氨酸)固定去细胞瓣在组织工程心脏瓣膜(TEHV)构建中的作用。方法酶+去污剂法制备去细胞瓣支架,在耦联剂 Sulfo-LC-SPDP 作用下甘-精-甘-天冬-丝-脯-半胱氨酸(GRGDSPC)肽与之化学结合,然后在其表面种植大鼠肌成纤维细胞,构建 TEHV。体外培养2周后取出瓣叶,作 HE 染色和扫描电镜观察,检测羟脯氨酸反映胶原含量,检测 DNA 反映细胞增殖,逆转录-聚合酶链反应(RT-PCR)方法检测Ⅰ型胶原、赖氨酰氧化酶(LOX)mRNA的表达。结果与对照组比较,经肽固定的实验组瓣膜细胞密集且组织致密,羟脯氨酸和 DNA 含量增高,Ⅰ型胶原、LOX mRNA表达增强。结论采用 RGD 肽表面修饰去细胞瓣,不仅可显著提高支架黏附性,而且可促进细胞生长和胶原分泌,有利于 TEHV的构建。

Objective To investigate effects of the RGD （arginine-glycine-aspartic acid） containing peptides on tissue engineering heart valves （TEHV）. Methods Decelluarized valve scaffolds, prepared by trypsinase and Triton-100, were immobilized with GRGDSPC peptide with the help of a coupling reagent Sulfo-LC-SPDP, and then were seeded by myofibroblasts harvested from rats. After culture in vitro for two weeks, light and electron microscopical observation was performed. DNA assay for cell amount and hydroxyproline content for collagen production were measured. RT-PCR technique was used to analyze expressions of type Ⅰ collagen and lysyl oxidase mRNA. Results Compared to untreated decelluarized valve scaffolds, histological investigation showed that cells and extracellular matrix were more flourishing. DNA assay, hydroxyproline content, and expression of type Ⅰ collagen and lysyl oxidase-mRNA were also increased. Conclusion It is an effective method for decellularized valve scaffold coupled with RGD peptides to improve cell adhesion, cell proliferation and collagen growth, which is helpful to construct tissue engineering heart valves.