骨髓源内皮祖细胞分泌的外泌体对大鼠创伤性皮肤缺损修复的促进作用

发布时间：2018-03-10 07:42

本文选题：内皮祖细胞　切入点：外泌体　出处：《吉林大学学报(医学版)》2017年04期 　论文类型：期刊论文

【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(P0.05)。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(P0.05)。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。
[Abstract]:Objective: to investigate the effect of exocrine EPCs-Exoses secreted by endothelial progenitor cells from bone marrow on angiogenesis and collagen deposition, and to elucidate the possible mechanism of promoting skin wound healing. EPCs-Exos.EPCs-Exos.EPCs-Exos.EPCs-Exos.EPCs-Exos.EPCs-Exos.EPCs-Exos.EPCs-Exos.EPCs-Exos.EPCs-Exos.EPCs-Exos.EPCs-Ex@@. The same amount of PBS50 and 150mg 路L ~ (-1) EPCs-Exos were injected into the area around the skin defect of the 4 groups respectively. The wound closed area of the rats in each group was measured on the 7th and 14th day, and the tissue of the healing area was taken for Masson trichrome staining and CD31 immunofluorescence staining. To evaluate the therapeutic effect of EPCs-Exos, the EPCs-Exos medium containing PBS50 and 150 mg 路L ~ (-1) was co-cultured with human umbilical vein endothelial cells (HUVECs) in vitro. The migration of HUVECs and the formation of capillary network were detected by scratch test and tube forming test. The expression of vascular endothelial growth factor (VEGF) in HUVECs was detected by Western blotting method. Results: proto was isolated and identified successfully. The CD31 immunofluorescence staining of EPCs was positive for both DiL-ac-LDL and FITC-UEA-I. The morphology of EPCs-Exos was nearly round by electron microscope. In the skin injury model of rats injected with EPCs-Exos, the diameter of scar of skin defect gradually decreased and the degree of scar healing increased with the increase of injection dose. The difference between the two groups was statistically significant (P 0.05). EPCs-Exos promoted the healing of collagen in a dose-dependent manner. 150 mg 路L ~ (-1) EPCs-Exos injection group was the most obvious at 14 days after maturation. In vitro, EPCs-Exos stimulated endothelial progenitor cells in a dose-dependent manner to promote their migration and hair. The formation of fine vascular network and its up-regulation of migration rate of VEGFA expression level of 150 mg 路L ~ (-1) EPCs-Exos group. The number of reticulum branches and the expression of VEGFA were higher than those of 50 mg 路L ~ (-1) EPCs-Exos and PBS groups (P 0.05). Conclusion: EPCs-Exos can promote the repair of traumatic skin defects by positively regulating the function of vascular endothelial cells in rats.
【作者单位】：锦州医科大学附属第一医院骨科;辽宁省抚顺市第二医院烧伤整形外科;
【基金】：国家自然科学基金资助课题(81572140)
【分类号】：R641

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