3D生物打印构建HGFs和HUVECs共培养体系促进HUVECs成血管  

3D Bioprinting of HGFs and HUVECs Co-culture System Promotes Vascularization of HUVECs

作　　者：李俊俊 王雯 郭慧颖 袁长永 夏廷旭 王鹏来 LI Junjun;WANG Wen;GUO Huiying;YUAN Changyong;XIA Tingxu;WANG Penglai(School of Stomatology,Xuzhou Medical University,Xuzhou 221004,China;Department of Oral Implantology,Affiliated Stomatological Hospital of Xuzhou Medical University,Xuzhou 221000,China.)

机构地区：[1]徐州医科大学口腔医学院,江苏徐州221004 [2]徐州医科大学附属口腔医院种植科,江苏徐州221000

出　　处：《口腔医学研究》2024年第4期297-303,共7页Journal of Oral Science Research

基　　金：国家自然科学基金项目(编号:82201071);江苏省研究生科研与实践创新计划项目(编号:KYCX23_2949)。

摘　　要：目的:构建人牙龈成纤维细胞(human gingival fibroblasts,HGFs)和人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)共培养体系,促进HUVECs成血管。方法:取HGFs和HUVECs培养传代至3~5代进行实验。通过慢病毒转染用红色荧光蛋白标记HUVECs。构建HGFs和HUVECs二维共培养体系,将HGFs与HUVECs以不同比例(4∶1、1∶1、1∶4)共培养,荧光显微镜下观察血管网络形成情况。配制5%甲基丙烯酰化明胶(GelMA)30胶,构建HGFs和HUVECs三维共培养体系,激光共聚焦显微镜下观察血管网络的形成情况。对HUVECs形成的血管网络进行定量分析。将HUVECs从三维共培养系统中分离出来,评估单独培养和共培养一定时间后HUVECs的增殖、迁移和小管形成效果以及相关血管生成基因的表达水平。结果:二维共培养时,当HGFs和HUVECs以比例1∶1共培养时血管网络形成的效果最佳。三维共培养时,HGFs可以促进HUVECs血管生成。HGFs和HUVECs共培养组的增殖、迁移和小管形成效果更好,相关血管生成基因的表达水平远远高于HUVECs单独三维培养组。结论:HGFs和HUVECs共培养能够引导和促进HUVECs出芽及迁移。Objective:To construct a co-culture system of human gingival fibroblasts(HGFs)and human umbilical vein endothelial cells(HUVECs)to promote vascularization of HUVECs.Methods:HGFs and HUVECs were cultured to 3 to 5 passages for experiments.The HUVECs was labeled with a red fluorescent protein by lentiviral transfection.A two-dimensional co-culture system of HGFs and HUVECs was constructed.HGFs and HUVECs were co-cultured at different ratios(4∶1,1∶1,and 1∶4),and the formation of vascular network was observed under fluorescence microscope.5%methacrylylated gelatin(GelMA)30 hydrogel was prepared to construct a three-dimensional co-culture system of HGFs&HUVECs,and the formation of vascular network was observed under laser confocal microscope and quantitative analyzed.HUVECs were separated from three-dimensional co-culture system to assess the proliferation,migration,tube formation,and expression levels of relevant angiogenesis genes after single-culture and co-culture for a certain period.Results:In two-dimensional co-culture,vascular network formation was best when HGFs and HUVECs were cocultured in a ratio of 1∶1.In three-dimensional co-culture,HGFs can promote HUVECs angiogenesis.The co-culture of HGFs&HUVECs had better effects on proliferation,migration,and tube formation compared to the three-dimensional culture of HUVECs alone,and the expression levels of relevant angiogenesis genes were significantly higher.Conclusion:Co-culture of HGFs and HUVECs can guide and promote HUVECs migration and sprouting.

关 键 词：3D生物打印 成血管 组织工程 人脐静脉内皮细胞 人牙龈成纤维细胞 

分 类 号：R318[医药卫生—生物医学工程]