胆脂瘤基质成纤维细胞外泌体对血管新生的影响

Effect of exosomes derived from cholesteatoma perimatrix fibroblasts on angiogenesis in endothelial cells

目的探讨胆脂瘤基质的成纤维细胞分泌的外泌体(h CPFs-Exo)对血管新生影响。方法从5例胆脂瘤患者手术中获取中耳胆脂瘤及耳后皮肤标本各5份,分析胆脂瘤MRI表现,CD34免疫组化染色比较胆脂瘤基质下血管分布。培养胆脂瘤成纤维细胞(CPF),提取其分泌的外泌体,并使用投射电子显微镜及免疫印迹法鉴定。h CPFs-Exo作用于人脐静脉血管内皮细胞系(HUVECs),用迁移实验和成管实验分别检测其迁移和成管能力,实时定量PCR检测其血管新生基因的表达。结果胆脂瘤MRI表现为T1WI低信号,T2WI高信号,增强后边缘强化。CD34免疫组化染色示胆脂瘤基质血管密度与耳后皮肤存在显著性差异(P <0. 05)。投射电子显微镜观察h CPFs-Exo为直径30-100 nm囊性微泡,免疫印迹检测其表达外泌体标志性蛋白CD81、CD63、HSP70、TSG101和CD9。Dil标记的h CPFs-Exo可被HUVECs摄取。h CPFs-Exo干预后,HUVECs迁移能力增强,与未加外泌体的对照组相比,血管新生基因Flk1,Fli1,Angpt1,Ephrin,Tie2和Angpt2的表达水平明显上调(P <0. 05)。成管实验显示,h CPFs-Exo干预后HUVECs管状结构的长度显著性增加(P <0. 05)。结论 h CPFs-Exo具有促血管内皮细胞及血管新生作用。

Objective To evaluate the role of exosomes derived from human cholesteatoma perimatrix fibroblasts( h CPFs-Exo) in the angiogenesis. Methods Cholesteatoma tissues and normal postauricular skin specimens were collected from five patients with cholesteatoma undergoing tympynoplasty. MRI and immunohistochemical staining were used to investigate the vascularization of cholesteatoma. Exosomes from cholesteatoma primary fibroblasts( CPFs-Exo) were isolated,and Western blot assay and transmission electron microscope were used to identify the characteristic of the exosome. Expression of pro-angiogenic genes Flk1,Fli1,Angpt1,Ephrin,Tie2,and Angpt2 was evaluated by qRT-PCR. The role of CPFs-Exo in angiogenesis was studied by cell migration assay and tube formation assay. Results Low signal on T1 WI,high signal on T2 WI with the rim of enhancement on delayed gadolinium-enhanced T1 WI were found in patients with cholesteatoma. Immunohistochemical staining of CD34 further demonstrated the significant difference of density of blood vessels between cholesteatoma and postauricular skin( P < 0. 05). CPFs-Exo were 30-100 nm round and membrane-bound vesicles under TEM with positive expression of CD81,CD63,HSP70,TSG101,and CD9 by Western blot analysis. Dillabelled exosomes were transferred into human umbilical vein endothelial cells( HUVECs). After treatment with h CPFs-Exo,cell migration increased in HUVECs and expression of pro-angiogenic genes Flk1,Fli1,Angpt1,Ephrin,Tie2,and Angpt2 was upregulated.Tube formation by HUVECs was also significantly enhanced in the presence of h CPFs-Exo( P < 0. 05). Conclusion Exosomes derived from cholesteatoma perimatrix fibroblasts can promote angiogenesis in endothelial cells.