3D生物打印构建HGFs和HUVECs共培养体系促进HUVECs成血管

doi:10.13701/j.cnki.kqyxyj.2024.04.004

摘要/Abstract

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。

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

Abstract: 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.

Key words: 3D bioprinting, vascularization, tissue engineering, human umbilical vein endothelial cells, human gingival fibroblasts

李俊俊, 王雯, 郭慧颖, 袁长永, 夏廷旭, 王鹏来. 3D生物打印构建HGFs和HUVECs共培养体系促进HUVECs成血管[J]. 口腔医学研究, 2024, 40(4): 297-303.

LI Junjun, WANG Wen, GUO Huiying, YUAN Changyong, XIA Tingxu, WANG Penglai. 3D Bioprinting of HGFs and HUVECs Co-culture System Promotes Vascularization of HUVECs.[J]. Journal of Oral Science Research, 2024, 40(4): 297-303.

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