文献类型：期刊文献

中文题名：三维预血管化微组织中成纤维细胞促进内皮细胞出芽及迁移的研究

英文题名：Effect of fibroblasts on promoting the sprout and migration of endothelial cells in threedimensional pre-vascularized microstructures

作者：唐珺[1];谈金女[1];叶朝阳[1];周燕[1];谭文松[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室,上海200237

年份：2022

卷号：36

期号：7

起止页码：881

中文期刊名：中国修复重建外科杂志

外文期刊名：Chinese Journal of Reparative and Reconstructive Surgery

收录：CSTPCD;;Scopus;北大核心:【北大核心2020】；CSCD:【CSCD2021_2022】；PubMed;

语种：中文

中文关键词：组织工程;微组织;预血管化;转瓶动态培养;三维共培养

外文关键词：Tissue engineering;microstructures;pre-vascularization;rotating bottle dynamic culture;three-dimensional co-culture

摘要：目的 构建三维预血管化微组织,探索三维共培养体系中人成纤维细胞(human fibroblasts,HFs)对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)出芽和迁移的促进作用。方法 取HUVECs和HFs培养传代至3~5代进行实验。在二维共培养体系中,将HFs行PKH26染色且固定细胞密度后,与HUVECs以不同比例(1∶4、1∶1、4∶1)以及不同接种方式(两种细胞间隔48 h顺序接种、直接混合接种)共培养,荧光显微镜下观察血管网络形成情况。构建三维共培养体系,通过慢病毒转染分别用绿色和红色荧光蛋白标记HUVECs和HFs,将标记细胞接种于多孔微载体上,采用转瓶动态培养,分别制备HF微组织、HUVEC微组织、HF-EC微组织和HF&EC微组织;低渗结晶紫染色法观测微组织中细胞生长情况。将各微组织包埋于纤维蛋白凝胶中,激光共聚焦显微镜下观察HF微组织和HUVEC微组织中细胞生长贴附情况,4种微组织出芽情况以及HF-EC微组织、HF&EC微组织芽体细胞组成、出芽数量及芽体长度。制备HFs条件培养基,观察其对HUVEC微组织出芽的影响,以正常培养基培养作为对照。结果 二维共培养时HFs预培养有助于血管网状结构形成与稳定,且当两种细胞比例为1∶1时效果最佳。三维共培养时各微组织上细胞生长良好,HUVEC微组织无出芽,HF微组织、HF-EC微组织、HF&EC微组织均能出芽,具备良好血管化能力。HF-EC微组织芽体长度及出芽数目均优于HF&EC微组织(P<0.05)。共培养微组织中HF微组织先于HUVEC微组织出芽,两者出芽轨迹一致,芽体由HFs和HUVECs组成。在HFs条件培养基中,HUVEC微组织也能出芽。结论 在转瓶中按1∶1比例顺序接种HFs和HUVECs可制备HF-HUVEC预血管化微组织,且在三维共培养中HFs能够引导和促进HUVECs出芽及迁移。
Objective To construct three-dimensional(3D) pre-vascularized microstructures and explore the promoting effect of human fibroblasts(HFs) on the sprout and migration of human umbilical vein endothelial cells(HUVECs) in 3D co-culture system.Methods HUVECs and HFs were cultured and the 3rd to 5th generation cells were selected for subsequent experiments.In 2D co-culture system,HFs were stained with PKH26 and the cell density was fixed,which co-cultured with HUVECs in different ratios(1∶4,1∶1,4∶1) and inoculation methods(HUVECs inoculation at 48 hours after HFs,direct mixed inoculation).Then the formation of vascular like structures was observed under fluorescence microscope.In 3D co-culture system,HUVECs and HFs were labeled with green fluorescent protein and red fluorescent protein by lentivirus transfection,respectively.They were inoculated on porous micro-carriers followed by dynamically culturing in rotating bottles to prepare HF,HUVEC,HF-EC,or HF&EC microstructures.The cell growth in microstructures was testing by low permeability crystal violet staining.Subsequently,the microstructures were embedded in fibrin gel and the cell growth and adhesion in HF and HUVEC microstructures were observed by laser confocal microscopy.Laser confocal microscope were also used to observe the sprouts of 4 kinds of microstructures,as well as the cell composition,the number and length of sprouts from HF-EC and HF&EC microstructures.HFs conditioned medium was prepared to observe its effect on sprouts of HUVEC microstructures with DMEM as control group.Results In 2D co-culture system,HFs pre-culturing was helpful to the formation and stability of vascular like structures,and the best effect was when the ratio of two kinds of cells was 1∶1.In 3D co-culture system,it was found that the cells grew well on micro-carriers and had the ability of pre-vascularization.HUVEC microstructures did not sprout,but HF,HF-EC,and HF&EC microstructures could which indicated a good vascularization ability.The HF-EC microstructures were superior to HF&EC microstructures in terms of sprouts length and number(P<0.05).The tubes sprouting from cocultured group were composed of HFs and HUVECs,and HF microstructures migration preceded HUVEC microstructures always,and their migration trajectories were the same.HUVEC microstructures could sprout when cultured in HFs conditioned media.Conclusion HF-HUVEC pre-vascularized microstructures can be prepared by preculturing HFs before HUVECs and with the cell ratio at 1∶1 in a rotating bottle.In 3D co-culture system,HFs can promote and guide the sprout of HUVECs.