与胶原浮胶细胞共培养中内皮细胞血管新生相关基因的表达  

Expression of angiogenesis related genes in human umbilical vein endothelial cells within the floating gel co-culture system

作　　者：刘水[1] 李晓聪[1] 方宁涛[1] 谢尚喆[1] 潘銮凤[1] 

机构地区：[1]复旦大学上海医学院分子生物学实验室,上海市200032

出　　处：《中国组织工程研究与临床康复》2008年第42期8216-8220,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：上海市科委基金资助重点项目(04JC14012)~~

摘　　要：背景:内皮细胞和平滑肌细胞的相互调节是稳定血管壁结构的关键因素,两者共同培养能够分别影响各自的形态和功能。目的:利用Ⅰ型胶原浮胶建立人脐静脉内皮细胞与人血管平滑肌细胞共培养体系,分析细胞之间的相互作用。设计、时间及地点:单一样本观察,于2006-10/2008-01在复旦大学上海医学院分子生物学实验室完成。材料:新鲜脐带取自国际第一妇婴保健医院,产妇知情同意。方法:取新鲜脐带,利用胶原酶灌注法分离人脐静脉内皮细胞,组织块培养法分离人血管平滑肌细胞。人血管平滑肌细胞直接种植于培养板表面,人脐静脉内皮细胞种植于鼠尾Ⅰ型胶原表面制成胶原浮胶,并与培养板上的人血管平滑肌细胞共培养。以人脐静脉内皮细胞单独在浮胶底面培养,培养板表面无人血管平滑肌细胞为对照。主要观察指标:①免疫荧光方法鉴定人血管平滑肌细胞和人脐静脉内皮细胞。光镜和电镜观察人脐静脉内皮细胞形态。②反转录-聚合酶链反应进行基因表达分析。结果:原代培养的内皮细胞呈铺路石样形态,CD31染色阳性。原代培养的人血管平滑肌细胞免疫荧光染色α-SMA呈阳性。胶原支架上内皮细胞伸展,胶原纤维重新排列,24h后出现管腔样结构。在共培养体系中,人脐静脉内皮细胞的金属蛋白酶1和金属蛋白酶9基因表达量显著高于单独培养组人脐静脉内皮细胞的表达量(P<0.05)。但层粘蛋白γ2和组织因子途径抑制物2的基因相对表达量在两组间未见明显变化。结论:实验建立的共培养体系,可以观察到与平滑肌细胞共培养的血管内皮细胞形成更加丰富的网络状管腔样结构,更易于血管结构的形成。BACKGROUND： The inter-regulation between endothelial cells and smooth muscle cells is a basis of stabilizing the structure of vascular wall, and the co-culture of the two may influence each other＇s morphology and function. OBJECTIVE： A novel co-culture system as I collagen floating gel was explored to study interaction of human umbilicai vein endothelial cells （HUVECs） and human vascular smooth muscle cells （hSMCs）. DESIGN, TIME AND SETTING： A single sample observation was performed in the Laboratory of Molecular Biology, Shanghai Medical College of Fudan University （Shanghai, China） from October 2006 to January 2008. MATERIALS： Fresh umbilical cord was provided by Shanghai First Women and Children Health Care Hospital with the informed consent from the puerperant. METHODES： HUVECs and hSMCs were isolated using collagenase perfusion and tissue mass cell culture methods, respectively. Using rat tail type I collagen as the scaffold, we cultured a single layer of HUVECs on the downside of floating gel and co-cultured with a single layer of hSMCs attaching on the surface of cell culture plate. The crosstalk between HUVECs and hSMCs was allowed by culture media between these two cell layers, while culture plated without hSMCs were taken as controls. MAIN OUTCOME MEASURES： HUVECs and hSMCs were confirmed by immunofluorescent staining. The morphology of HUVECs was observed under light microscope and electron microscope. The gene expressions were analyzed by reverse transcription-polymerase chain reaction. RESULTS： Primary HUVECs showed typical cobblestone morphology and were confirmed by CD31 expression. Primary hSMCs were confirmed by α-SMA expression. When endothelial cells were cultured on the collagen floating gel, reorganization of collagen fibers was also observed in the surface of the gel and endothelial cells formed blood vessel-like network structure 24 hours later. The HUVECs-hSMCs interactions lead to a significantly increased expression of metailoprotease-1 and metalloprotease-9

关 键 词：脐静脉 内皮细胞 肌细胞 平滑肌 胶原Ⅰ型 基因表达 细胞培养 组织构建 

分 类 号：R392[医药卫生—免疫学]