自聚肽承载的Ephrin-b2/Fc重组蛋白在心肌梗死治疗中的缓释效应

Controlled Release of Recombinant Ephrin-b2/Fc from Self Assembling-peptide in Myocardial Infarction Therapy

目的通过体外观测和体内实验探讨自聚肽RAD16-Ⅱ对Ephrin-b2/Fc重组蛋白的控制释放效应及其免疫原性,解决心肌梗死蛋白质治疗中裸露的蛋白质在机体有效作用时间短且易被降解的问题。方法体外观测RAD16-Ⅱ的塑型及对Ephrin-b2/Fc重组蛋白的控制释放;构建SD大鼠心肌梗死模型,将存活大鼠分为2组分别予心肌内注射Ephrin-b2/Fc蛋白(E组,n=25)和自聚肽Ephrin-b2/Fc蛋白凝胶(ES组,n=25),在设定的时间点(1 h,3h,24 h,7 d,14 d)各收集心肌组织和血清样本5个,分别用免疫荧光和免疫印迹技术检测eprhin-b2蛋白驻留和丢失情况;皮下注射RAD16-Ⅱ,5 W后ELISA法检测血清抗体滴度,评估RAD16-Ⅱ的免疫原性。结果 RAD16-Ⅱ在PBS中可自我聚合组装成纳米纤维网状结构;这种结构在体外可使Ephrin-b2/Fc重组蛋白的释放持续144 h,其中超过50%量在120 h内释放;免疫荧光显示除注射后1 h外,Ephrin-b2/Fc蛋白在ES组的驻留量明显高于同期的E组(P<0.05);免疫印迹显示注射早期,ES组Ephrin-b2/Fc蛋白的血液释入量明显少于同期的E组(P<0.05);ELISA法检测血清抗RAD16-Ⅱ抗体效价与阴性对照组无显著差异。结论 RAD16-Ⅱ可明显地延缓Ephrin-b2/Fc重组蛋白的释放,是承载Ephrin-b2/Fc蛋白用于心肌梗死治疗和缺血性研究的较为可靠和安全的生物载体材料。

Aim To explore the controlled release effect of the recombinant Ephrin-b2 /Fc chimera from self assembling peptide of RAD16-Ⅱ and the immunogenicity of RAD16-Ⅱ. Take RAD16-Ⅱas a delivery vehicle to prevent the ungroomed Ephrin-b2 / Fc protein from being degraded in vivo and increase therapeutic effect. Methods The moulding of the oligopeptide was observed by macroscopy and microscopy. The releasing effect was assayed by releasing curve obtained from test in vitro. In vivo,Sprague-Dawley rats were prepared to acute myocardial infarction models by ligation of the left anterior descending branch. The survival rats were divided into 2 groups and separately intra myocardially injected recombinant Ephrin-b2 / Fc proteins( E group,n = 25) and sapeptide- Ephrin-b2 / Fc protein complex( ES group,n = 25). At set time( 1 h,3 h,24 h,7 d,14 d),the samples of the myocardial tissue and sera were collected and respectively used for immunofluorescence and westernblot to determine the remaining or losing of the protein. RAD16-Ⅱsolution was administrated through subcutaneous injection. 5 weeks later,the titer of anti-sapeptide in serum was assayed by ELISA. Results It is observed that RAD16-Ⅱsolution can self-assemble into nanofibers which fabricate net structure in condition of PBS. Within net structure,the releasing duration of chimeric Ephrin-b2 / Fc lasted up to 144 h and more than 50% mass was released in 120 h. As shown in immunofluorescence,the retention duration of the chimeric protein in ES group was significantly longer than that in E group at the same time( P < 0. 05). And westernbolt also demonstrated the amount of protein releasly into blood in ES group was less significant than that in E group( P < 0. 05). The titer of the antibody was not significantly different from RAD16-Ⅱ administration to negative control group,which showed that RAD16-Ⅱ would not trigger immune response. Conclusion RAD16-Ⅱ oligopeptide can postpone the release of chimeric Ephrin-b2 / Fc both in vivo and in vitro. It is a relatively reliable and safe biomaterial delivery vehicle of Ephrinb2 / Fc protein in myocardial infarction and ischemia research.