摘要目的 利用噬菌体展示技术,在心肌活体切片上筛选能特异性结合心肌的靶向短肽,提高药物心肌组织靶向运输效率,为杜兴肌肉萎缩症(DMD)及其他心肌相关疾病的靶向治疗提供有效的候选靶向肽.方法 制备心肌活体切片,体外培养心肌活体切片并用免疫组化技术检测其蛋白活性.将噬菌体文库以1x1012pfu的滴度与体外培养的心肌活体切片共孵育,回收并扩增与心肌活体切片结合的噬菌体,再经5轮体外生物淘选获得特异性靶向心肌的噬菌体,测序分析候选噬菌体的插入序列并对高效富集的噬菌体进行体内组织分布验证.结果 成功建立了心肌活体切片体外培养平台,获得了具有良好生物学活性的心肌活体切片,活体切片培养48 h后,仍能检测到抗肌萎缩蛋白的正常表达和定位.在此基础上,利用噬菌体文库对心肌活体切片进行5轮筛选,回收滴度测定结果显示淘选具有良好的富集效应.筛选后的噬菌体测序分析和体内组织分布验证结果显示,候选噬菌体在心肌、骨骼肌中出现明显富集,而在肝、肾组织中分布显著降低.结论 筛选出的候选噬菌体在心肌和骨骼肌中均表现为较高的结合效率,证明候选短肽具有心肌靶向能力,同时也为DMD的心肌靶向治疗提供了一种新方法.

abstractsObjective To screen cardiac-specific short-acting peptides on live myocardial slices using phage display technology,so as to improve the targeted delivery efficiency of drugs in myocardium and provide effective candidates for the targeted therapy of Duchenne muscular dystrophy (DMD) and other cardiomyopathies.Methods Myocardial tissue slices were prepared and cultured in vitro.The protein activities of the tissues were examined by immunohistochemistry.The in vitro cultured myocardial tissue slices were co-incubated with phage library (1×1012 pfu),and the phages that bound to the myocardium were recovered and amplified.The cardiac-specific targeting phages were identified by five rounds of in vitro phage biopanning.The candidate phage-related insertion sequence was sequenced,and the in vivo tissue distribution of the highly enriched phages was verified.Results A platform for in vitro culturing of live myocardial slices was established.Myocardial slices with good biological activity were obtained.After 48 hours of culturing,the normal expression and localization of Dystrophin protein were detected.Using phage library,candidate phages were screened after five rounds of phage biopanning.The results of the sequencing analyses and in vivo tissue distribution verification indicated that the selected candidate phages showed significant enrichment in myocardium and skeletal muscle,and showed low levels in liver and kidney tissues.Conclusions The candidate phages showed higher binding efficiency in both myocardium and skeletal muscle,indicating that the candidate peptides had myocardial targeting property,and that can provide a new method for myocardial targeting therapy of DMD.