尿源性干细胞的外泌体修复骨不连的作用研究

Role of exosomes of urine-derived stem cells in nonunion repair

目的评估尿源性干细胞(USC)的外泌体(Exos)能否促进骨不连的愈合,并探讨其作用机制。方法从人尿液中提取并培养USC,采用超滤法提取USC来源的Exos(USC-Exos)用于研究。应用透射电子显微镜检测USC-Exos的形态和粒径,应用QNano平台检测USC-Exos的大小和密度。体外实验:取新鲜分离的USC-Exos悬液,按照不同密度0、107、108个/mL分别配制新的含有USC-Exos的人脐静脉内皮细胞(HuVECs)完全培养基,对应分组为对照组、USC-Exos 107组、USC-Exos 108组。进行CCK-8细胞增殖实验、Transwell迁移实验、成管实验,验证USC-Exos促进HuVECs增殖、迁移、成管作用。体内实验:构建大鼠股骨骨不连的动物模型,将30只大鼠随机分为空白组(向骨不连周围注射500μL PBS)、不含USC-Exos的纯光致亚胺交联凝胶(piGEL)组(piGEL组,向骨不连周围注射500μL piGEL)、含有109/mL USC-Exos的USC-Exos-piGEL组(USC-Exos-piGEL组,向骨不连周围注射500μL USC-Exos-piGEL复合体),每组10只。分别于术后第4、8周行X线摄片、micro-CT检查和组织学检查,术后第8周对组织切片进行免疫荧光检查,观察愈合情况和骨不连周围血管再生情况。结果 USC-Exos直径约80 nm,形态呈类球形,有完整的膜结构,Exos悬液包含的USC-Exos约为1.97×1010个/mL,直径90~160 nm。CCK-8增殖实验:细胞培养第4、5、6天,USC-Exos 107组和USC-Exos 108组HuVECs的波长450 nm处吸光度(A450)值均显著高于对照组同时间(P值均<0.05),USC-Exos 108组HuVECs的A450值显著高于USC-Exos 107组同时间(P值均<0.05)。Transwell迁移实验:USC-Exos 107组和USC-Exos 108组完成迁移的细胞数均显著多于对照组(P值均<0.05),USC-Exos 108组完成迁移的细胞数显著多于USC-Exos 107组(P<0.05)。成管实验:USC-Exos 107组和USC-Exos 108组的分支点均显著多于对照组(P值均<0.05),小管长度均显著长于对照组(P值均<0.05);USC-Exos 108组的分支点显著多于USC-Exos 107组(P<0.05),小管长度显著长于USC-Exos 107组(P<0.05)。影像学检查:USC-Exos-piGEL组在术后4周时,骨不连未修复,有骨痂形成,但仍有骨皮质不连续,骨缺损未完全愈合;第8周时,骨不连得到修复,骨皮质连续。USC-Exos-piGEL组术后第4、8周骨不连处的骨体积分数均显著大于空白组和piGEL组(P值均<0.05)。组织学检查:USC-Exos-piGEL组骨不连断端骨皮质连续,新生的骨组织细胞排列尚不整齐,与正常骨组织间有明显的分界,细胞以骨细胞为主,少见骨髓细胞。免疫荧光检查:USC-Exos-piGEL组的血管内皮细胞较多,分布较密,围成的闭合管样结构完整,表明血管生成量较多。结论 USC-Exos能够促进大鼠股骨骨不连的愈合,其修复作用可能与促进骨不连周围血管再生有关。

Objective To explore whether exosomes of urine-derived stem cells(USCs)can promote the healing of nonunion and its mechanism.Methods Human USCs were extracted and cultured,and secreted exosomes(USC-Exos)were extracted by ultrafiltration.Particle size and concentration of USC-Exos were determined by transmission electron microscopy and QNano techniques.Cell counting kit-(CCK)-8 cell proliferation assay,Transwell test and tube-forming experiment were used to verify the proliferation,migration and tube formation of human umbilical vein endothelial cells(HuVECs),which were cocultured at 0,107,and 108/mL,respectively.An animal model of femoral nonunion was constructed in rats.Then 30 rats were randomly divided into 3 groups(n=10):PBS group(500μL PBS were injected around the tissues of nonunion),piGEL group(500μL piGEL were injected)and USC-Exos-piGEL group(500μL USC-Exos-piGEL containing 109/mL USC-Exos were injected).Radiography,micro-CT and histological examination were applied at the 4th and 8th week after operation.Immunofluorescence was used to assess the healing of nonunion and the regeneration of blood vessels around the nonunion.Results USC-Exos was spherical in shape and had a complete membrane structure,with a diameter of 80 nm.The concentration of exosome suspension was about 1.97×1010 cells/mL.In CCK-8 cell proliferation assay,Transwell test and tube-forming experiment,USC-Exos showed a good promotion effect on HuVECs,which was positively correlated with the content of USC-Exos(P<0.05).Imageological examination showed that bony callus had formed but bone cortex was incontinuous at the 4th after operation;bone nonunion was repaired and bone cortex was continuous at the 8th after operation.The ratio of bone volume fraction in USC-Exos-piGEL group was significantly larger than those in blank group and piGEL group at 4th and 8th after operation(all P<0.05).Histological examination showed that bone cortex at the nonunion became continuous;the arrangement of new bone tissue cells was not uniform and there was a clear demarcation between them and normal bone tissue;the cells were mainly osteocytes,but few bone marrow cells were found in USC-Exos-piGEL group.Immunofluorescence detection showed significant angiogenesis in the nonunion of USC-Exos-piGEL group.Conclusion USC-Exos can effectively repair nonunion,which may be related to the increase of vascular regeneration around the nonunion.