环状RGDfK肽对肌成纤维细胞整合素αVβ3表达调控的实验研究  

Cyclo-RGD Peptide Regulates the Expression of Integrin αVβ3 Gene of Myofibroblast on the Decellularized Scaffolds

作　　者：董毅[1] 董念国[1] 史嘉玮[1] 洪昊[1] 谢霆[1] 胡畅[1] 陈思[1] 邓诚[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院心外科,武汉430022

出　　处：《中国胸心血管外科临床杂志》2008年第5期360-364,共5页Chinese Journal of Clinical Thoracic and Cardiovascular Surgery

基　　金：国家自然科学基金资助项目(30571839;30600608)~~

摘　　要：目的探讨环状精氨酸-甘氨酸-天冬氨酸-D-脯氨酸-赖氨酸短肽(Cyclo-RGDfK)能否提高去细胞瓣对肌成纤维细胞(myofibroblast)的黏附性能,以及RGD肽对Integrin αVβ3基因表达的调控。方法组织块培养法获取大鼠主动脉壁肌成纤维细胞(myofibroblast),免疫荧光染色检测波形蛋白(Vimentin)和α-平滑肌肌动蛋白(α-SMA)分子在培养myofibroblast中的表达。将去细胞瓣随机分为3组(每组7个),A组:去细胞瓣未接受任何处理;B组:去细胞瓣与交联剂1-乙基-3-(二甲基氨基丙基)-碳化二亚胺盐酸盐(EDC)反应36h;实验组:去细胞瓣与EDC反应36h后,再与Cyclo-RGDfK反应24h,使RGD肽共价结合于去细胞瓣。将第5代myofibroblast分别接种至各组瓣膜上。HE染色和扫描电子显微镜观察细胞黏附增殖状况,半定量逆转录-聚合酶链反应(RT-PCR)检测Integrin αVβ3基因在各组的表达。结果免疫荧光染色显示原代培养的myofibroblast生长良好,高表达Vimentin和α-SMA分子。HE染色和扫描电子显微镜显示myofibroblast在实验组的生长好于A组和B组,半定量PCR示Integrin αVβ3 mRNA在实验组的表达高于A组和B组,差异有统计学意义(P<0.05),而A组与B组比较差异无统计学意义(P=0.900)。结论Cyclo-RGDfK促进myofibroblast在去细胞瓣上黏附,此效应可能与RGD肽上调Integrin αVβ3基因表达有关。Objective To observe whether Cyclo-RGDfK （Arg-Gly-Asp-D-Phe-Lys ） could enhance the adhesion of myofibroblast to decellularized scaffolds and upregulate the expression of Integrin αVβ3 gene. Methods Myofibroblast from the rat thoracic aorta was acquired by primary cell culture. The expression of Vimentin and a smooth muscle actin （α-SMA） has been detected by immunoflurescent labeling. Decellularized valves have been randomly divided into three groups （each n=7）. Group A （blank control）： valves do not receive any pretreatment; Group B： valves reacted with linking agent- N-Ethyl-N-（3-dimethylaminopropyl）-carbodiimide hydroehloride （EDC） for 36 hours before being seeded; Experimental group： Cyclo-RGD peptide has been covalently immobilized onto the surface of scaffolds by linking agent EDC. The fifth generation of myofibroblast has been planted on the scaffolds of each group. The adhesion of myofibroblast to the scaffolds was evaluated by HE staining and electron scanning microscope. The expression of Integrin αVβ3 was quantified by half-quantitative reverse transcription polymerase china reaction （RT-PCR）. Results We can see that myofibroblast has exhibited strong positive staining for Vimentin and a-SMA. Besides, it has been shown that the expression of Integrin αVβ3 was much higher in the experimental group than that of the group A and group B（P〈0.05）. There was no statistically difference in group A and group B （P= 0. 900）. Conclusion RGD pretreatment does enhance the adhesive effieiency of seeding cells to the scaffolds and this effect may be related to the upregulation of Integrin αVβ3.

关 键 词：组织工程瓣膜 去细胞瓣 精氨酸-苷氨酸-天冬氨酸肽 细胞黏附 整合素基因 半定量聚合 酶链反应 

分 类 号：R318.11[医药卫生—生物医学工程]