摘要
目的建立人脐动脉内皮细胞-平滑肌细胞共培养模型,以体外模拟人动脉壁,为动脉粥样硬化及炎症等疾病的发病机制和治疗研究打下基础。方法采用胶原酶灌注消化法从人脐动脉中原代培养获得人脐动脉内皮细胞(human umbilical artery endothelial cell,HUAEC),采用组织块贴块法从人脐动脉中原代培养获得人脐动脉平滑肌细胞(human umbilical artery smooth muscle cell,HUASMC)。用含有抗坏血酸(≥50μg/mL)的培养基孵育平滑肌细胞,使之合成并分泌胶原蛋白,形成内皮细胞的生长基质;然后将内皮细胞以饱和密度接种到平滑肌细胞上,使内皮细胞和平滑肌细胞直接接触并整合形成内皮细胞-平滑肌细胞共培养(EC-SMC co-culture)模型。分别用免疫荧光染色和Dil-Ac-LDL吞噬实验对共培养模型进行形态学和功能学的鉴定。结果形态学鉴定结果显示,两种细胞已成功整合,模拟出体内动脉壁的形态。Dil-Ac-LDL吞噬实验的结果显示共培养模型的内皮细胞中有荧光信号,并且共培养模型中内皮细胞Dil-Ac-LDL的内吞量显著高于单纯内皮细胞(EC monoculture or ECmonolayer),证明共培养模型中内皮细胞与平滑肌细胞已建立联系。结论本实验成功建立人脐动脉内皮细胞-平滑肌细胞共培养模型,可在体外更好地模拟人动脉壁形态和基本功能。
Objective To establish a human umbilical artery EC-SMC co-culture model, and mimic the morphological and functional characteristics of human arterial wall, for further reseach of the pathological mechanism and therapy of atherosclerosis and imflammatory damage. Methods We secceeded in the primary culture of human umbilical artery endothelial cells (HUAEC) and human umbilical artery smooth muscle cells (HUASMC) by collagenase perfusion digestion and tissue planting, respectively. HUASMCs were incubated in a medium with ascorbic acid at the concentration greater than 50 μg/mL to produce collagen, which was considered as the extracellular matrix for ECs. Then HUAECs were seeded directly upon HUASMCs in a saturate density for sufficient direct physical interaction between ECs and SMCs. The morphological characteristic of EC-SMC co-culture was identified by immunofluorescence staining, and the function of EC-SMC co-culture was identified by Dil Ac-LDL uptake test. Results The morphological identification showed that the entire surface of HUASMCs was covered by a confluent monolayer confluent monolayer, which indicated that the model had simulated the morphological characteristic of human arterial wall. The results of DiI-Ac-LDL uptake test showed that there was a fluorescent signal in HUAECs. Compared with EC monoculture, the Dil-Ac LDL uptake of HUAECs was increased significantly in the co-culture system. All the reseach results indicated that there was an interaction between HUAECs and HUASMCs in the co-culture system. Conclusions In the present study, human umbilical artery EC-SMC co-culture model was constructed successfully, which could mimic the morphological characteristic and basic functions of human arterial wall.
出处
《复旦学报(医学版)》
CAS
CSCD
北大核心
2009年第6期768-775,共8页
Fudan University Journal of Medical Sciences