负载干细胞外囊泡的缓释聚左旋乳酸微球在促肝细胞增殖中的作用

The effect of the extracellular vesicle loaded polylactic acid microspheres in promoting hepatocyte proliferation

目的制备一种调控释放干细胞外囊泡的微粒子递送系统,仅干预1次后长期发挥增强肝细胞增殖能力的作用。方法采用差速离心法提取干细胞外囊泡并鉴定,透射电镜观察外囊泡结构和免疫印记检测外囊泡膜标志蛋白,评价所提取外囊泡符合规范。采用乳液-溶剂挥发法制备“壳-核”结构聚左旋乳酸(poly-L-lactic acid,PLA)微球,在微球“核”结构位置负载干细胞外囊泡(extracellular vesicles,EVs)得到EVs-PLA微球。通过扫描、透射电镜检测微球及外囊泡形貌,粒径分析,缓释实验检测EVs-PLA微球缓释周期,扫描电镜检测缓释至8周EVs-PLA微球形貌变化;将EVs-PLA微球与肝实质细胞共培养,鬼笔环肽/DAPI染色评价EVs-PLA微球生物相容性和细胞毒性,CCK8评价EVs-PLA微球对细胞增殖活力的影响作用,免疫印记检测EVs-PLA微球共培养后细胞中增殖标志蛋白表达量并统计数据结果。结果对比EVs-PLA干细胞外囊泡微球处理组和对照组的肝实质细胞增殖能力评价,发现EVs-PLA微球未造成细胞凋亡和细胞结构发生变异,具有生物相容性无细胞毒性;EVs-PLA微球的“壳-核”结构调控所负载的干细胞外囊泡的释放速率和释放周期,EVs-PLA微球可缓慢释放EVs,单次干预后持续发挥促进肝实质细胞增殖的生物作用。结论EVs-PLA微球可缓慢释放EVs,单次干预后持续发挥增强肝细胞增殖能力的作用,为探索外囊泡促肝细胞增殖的微递送系统提供了新的思路。

Objective To prepare a microparticle delivery system that regulates the release rate of extracellular vesicles(EVs),and to exert long-term enhancement of liver cell proliferation after only one intervention.Methods EVs was extracted by differential centrifugation.The structure of the EVs was observed by transmission electron microscopy and the membrane marker protein of EVs was detected by Western blotting.EVs-PLA microspheres with"core-shell"structure were prepared by emulsion-solvent evaporation method.Scanning and transmission electron microscopy were used to detect the morphology of EVs-PLA microspheres and EVs.The release test detected the release behavior of EVs in EVs-PLA microspheres.Scanning electron microscopy was used to detect the morphological changes of EVs-PLA microspheres at 8 weeks of release.EVs-PLA microspheres were co-cultured with hepatocytes,and Phalloidin/DAPI staining was used to observe the cell morphology and evaluate the cytotoxicity of the microspheres.CCK8-test was used to evaluate the cell proliferation activity.Western blot analysis was used to detect extracellular vesicles membrane marker protein expression.Results Comparing the ability of hepatocyte proliferation in the group treated with EVs-PLA microspheres and the control group,it was found that EVs-PLA microspheres did not cause cell apoptosis and mutation in cell structure,had biocompatibility and no cytotoxicity.The EVs-PLA microspheres with"core-shell"structure regulated the release behavior of EVs,which can continuously release EVs,exerting a continuous biological role in promoting hepatocyte proliferation after a single intervention.Conclusions The EVs-PLA microspheres can control-release EVs and promote hepatocyte proliferation continuously after a single intervention,providing a reference for further exploration of EVs-loaded delivery systems in promoting liver regeneration.