自噬在hAMSCs促进血管新生作用的初探

hAMSCs enhance angiogenesis through autophagy

目的:通过构建人羊膜间充质干细胞(hAMSCs)与人脐静脉内皮细胞(hUVECs)3D共培养体系,探讨自噬与血管新生的相关性。方法:使用蛋白酶和胶原酶消化法及胶原酶消化法提取hAMSCs和hUVECs。通过流式细胞仪、免疫荧光及多向分化实验鉴定hAMSCs和hUVECs。通过3D成管实验观察0、3、6、12、24、48、72 h hAMSCs-hUVECs(共培养)组及hUVECs(单培养)组中hUVECs出芽的长度及面积,检测各时间点中反应自噬水平的蛋白ATG5、Beclin-1、LC3Ⅱ表达水平。结果:流式细胞仪检测发现,hAMSCs中间充质细胞表面分子标志呈阳性表达,造血干细胞及血管细胞相关的表面分子标志呈阴性表达;hUVECs中CD34呈阳性表达,CD45呈阴性表达。免疫荧光检测发现,hUVECs细胞膜上表达CD31,胞浆中表达抗血管性血友病因子(vWF)。多向分化实验证实hAMSCs具有向成骨细胞、成脂细胞及成软骨细胞分化的潜能。3D成管实验中共培养组在12 h以后出芽的长度及面积均高于单培养组,共培养组ATG5表达水平在3、6 h高于单培养组,共培养组Beclin-1表达水平在0、3、6 h高于单培养组,共培养组LC3 Ⅱ表达水平在0、3、6、12 h高于单培养组。结论:本研究发现hAMSCs可以促进血管新生,并证明其促进作用与共培养体系建立早期的胞内自噬水平增高密切相关。

Objective： To discuss the correlation of autophagy and angiogenic capacity in co-culture using human amniotic membrane-derived mesenchymal stem cells（hAMSCs） with human umbilical vein endothelial cells（hUVECs） in 3D gels by comparing to hUVECs-monoculture. Methods：To isolate hAMSCs and hUVECs,Amnion were incubated with dispase and collagenase,while umbilical veins were digested with collagenase type 1. Identification of hAMSCs and hUVECs through Flow Cytometry,immunofluorescence staining and Multilineage differentiation. The length of sprouting and average vessel area were surveyed in h UVEC-h AMSC coculture and h UVECs-monoculture at 0,3,6,12,24,48,72 h. ATG5,Beclin-1 and LC3Ⅱ expression was analyzed in co-culture and monoculture by western blotting a 0,3,6,12,24,48,72 h. Results： hAMSCs were positive for MSC markers,while negative for hematopoietic and vascular cellrelated markers. hUVECs were positive for CD34 and negative for CD45. hUVECs expressed CD31 on membrane surface and vWF in cytoplasm. The length of sprouting and average vessel area in co-culture were more than those in monoculture after 12 h. Expression of ATG5 in co-culture increased significantly in monoculture at 3 and 6 h,while Beclin 1 and LC3Ⅱ rose remarkably in h UVEC-h AMSC co-culture before 6 h and 12 h respectively. Conclusions： In conclusion,this study discovered that h AMSCs can promote angiogenic capacity and demonstrated that the capacity of promote angiogenic had intimate connection withelevated level of intracellular autophagy at early co-culture system.