摘要
目的:探讨骨髓源内皮祖细胞分泌的外泌体(EPCs-Exos)对血管新生和胶原沉积的作用,阐明其促进皮肤伤口愈合的可能机制。方法:分离、培养并鉴定内皮祖细胞(EPCs),同时分离并鉴定EPCs-Exos。观察分析EPCs摄取EPCs-Exos。16只大鼠随机分为4组,建立皮肤缺损模型,分别将等量的PBS,50、100和150mg·L^(-1) EPCs-Exos分别注射至4组大鼠的皮肤缺损周围区域。在0、3、7和14d测量各组大鼠伤口闭合区域的范围;14d时取愈合区组织进行Masson三色染色及CD31免疫荧光染色,评估EPCs-Exos的治疗效果。在体外,将含有PBS,50、100和150mg·L^(-1) EPCs-Exos培养基和人脐静脉内皮细胞(HUVECs)共培养,利用划痕实验和成管实验检测HUVECs的迁移和毛细血管网的形成。应用Western blotting法检测HUVECs中促血管生成相关基因血管内皮生长因子A(VEGFA)表达水平。结果:成功分离并鉴定原代EPCs,EPCs的CD31免疫荧光染色、DiL-ac-LDL和FITC-UEA-I双染均呈阳性。电镜观察EPCs-Exos形态为近圆形,直径为40~100nm。在注射EPCs-Exos的大鼠皮肤损伤模型中,随着注射剂量增大,皮肤缺损疤痕直径逐渐缩小,疤痕愈合度逐渐增加,组间比较差异有统计学意义(P<0.05)。EPCs-Exos呈剂量依赖性促进愈合皮肤组织胶原的成熟,14d时150mg·L^(-1) EPCs-Exos注射组最明显。在体外实验中,EPCs-Exos呈剂量依赖性刺激内皮祖细胞促进其迁移和毛细血管网的形成及上调VEGFA表达水平,150mg·L^(-1) EPCs-Exos组的迁移速率、网分支数目和VEGFA表达水平均高于50mg·L^(-1) EPCs-Exos组和PBS组(P<0.05)。结论:EPCs-Exos可以通过正向调控血管内皮细胞的功能而促进大鼠创伤性皮肤缺损的修复。
Objective： To explore the effects of exosomes derived from bone-marrow endothelial progenitor ceils （EPCs-Exos） on the angiogenesis and collagen deposition in vitro, and to illustrate the possible mechanism for EPCs-Exos to accelerate the cutaneous deficiency repair. Methods： The endothelial progenitor cells （EPCs） were isolated, cultivated and identified; at the same time, the EPCs-Exos were also isolated and identified. The EPCs-Exos uptake in EPCs was observed and analyzed. 16 rats were randomly divided into 4 groups, and then the models of skin defect were established, respectively. The equal volume of PBS and 50, 100, 150 mg · L^-1 EPCs-Exos were injected into the area around skin defect of the rats in 4 groups. The wound closed sizes on the 0, 3rd, 7th and 14th days were measured, and Masson＇ s trichrome staining and CD31 immunofluorescence staining were performed on the 14th day for evaluating the tissue healing efficacy after EPCs-Exos treatment. In vitro, the mediums containing PBS and 50, 100, 150 mg · L^-1 EPCs-Exos were used to culture the human umbilical vein endothelial cells （HUVECs）, respectively. Scratch test and tubule formation assay were used to detect the migration and capillary network formation of HUVECs. At the same time, Western blotting was used for analyzing the expression level of angiogenesis related gene vascular endothelial growth factor A （VEGFA） in HUVECs. Results： The primary EPCs were isolated and identified successfully, and EPCs-Exos were purified and characterized. The CD31 immunofluorescence staining and double staining of DiL-ac-LDL and FITC-UEA-I of EPCs were positive. The electron microscope results showed that EPCs-Exos were nearly spheroidal, with the diameter about 40--100 nm. For the models of rat skin injury treated by EPCs-Exos, with the increasing of injection doses, the sizes of skin defect scar were gradually reduced, the degrees of scar healings were gradually increased, and the differences between various groups were statistically significant （P~ 0.05）. EPCs-Exos promoted the collagen maturity of healing skin in a dose-dependent manner; on the 14th day, the effect in 150 mg ~ L-I EPCs-Exos group was the most significant. In vitro, EPCs-Exos promoted the migration and capillary network formation of HUVECs and increased the expression level of VEGFA; the migration rate, the net number of branches and the expression level of VEGFA in 150 mg· L^-1 EPCs-Exos group were significantly higher than those in 50 mg · L^-1 EPCs-Exos group and PBS group （P〈0.05）. Conclusion.. EPCs-Exos can promote the repair of traumatic skin defect of the rats by positively regulating the vascular endothelial cell function.
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2017年第4期672-678,I0001,I0002,共9页
Journal of Jilin University：Medicine Edition
基金
国家自然科学基金资助课题(81572140)
关键词
内皮祖细胞
外泌体
皮肤缺损
血管内皮生长因子A
endothelial progenitor cells
exosomes
cutaneous deficiency
vascular endothelial growth factor A
