嘌呤霉素介导的人真皮微血管内皮细胞的分离纯化培养与鉴定  

Puromycin-mediated Culture and Identification of Human Dermal Microvascular Endothelial Cells

作　　者：侯慧 李娇[1] 梁见楠 周玲[1] 梁艳阳 侯瑞霞[1] 李俊琴[1] 张开明[1] HOU Hui;LI Jiao;LIANG Jiannan;ZHOU Ling;LIANG Yanyang;HOU Ruixia;LI Junqin;ZHANG Kaiming(Department of Dermatology,Taiyuan Key Laboratory of Stem Cell for Immunological Dermatosis,Taiyuan Central Hospital,Taiyuan 030009,China)

机构地区：[1]免疫性皮肤病干细胞山西省重点实验室太原市中心医院皮肤科

出　　处：《中国皮肤性病学杂志》2019年第11期1255-1259,共5页The Chinese Journal of Dermatovenereology

基　　金：国家自然科学基金(81602768,81803146)

摘　　要：目的获取纯度较高的人真皮微血管内皮细胞(HDMEC)。方法取手术切除的多余包皮剪碎,通过两次酶消化接种于35 mm 6孔板中,加入含0.5μg/mL嘌呤霉素的内皮细胞培养基,3 d后更换新鲜培养基;采用倒置单光子共聚焦显微镜观察内皮细胞形态;流式细胞术检测HDMEC细胞表面标志CD31的表达情况。结果培养72 h后可见内皮细胞长出,细胞呈圆形,生长至第5天左右,以梭形细胞为主,第6~10天迅速增殖,形成比较紧密的融合细胞,细胞集落中心为圆形细胞,周围为梭形,呈放射状,第10天可达80%融合,细胞呈典型铺路石样,可见“漩涡状”特征。流式细胞术检测显示HDMEC的CD31表达阳性,CD44表达阴性,内皮细胞纯度达98%以上。结论该方法能够成功获得高纯度的人真皮微血管内皮细胞HDMEC,对皮肤疾病致病机制以及HDMEC的生物学特性和功能的深入研究具有重要意义。Objective To obtain highly purified human dermal microvascular endothelial cells(HDMEC).Methods Surgically removed superfluous foreskin was minced into pieces.Human dermal microvascular endothelial cells were obtained through enzymatic digestion twice and were incubated into 35 mm dish plates with endothelial growth medium containing 0.5μg/mL puromycin.The medium was renewed after three days.Morphology of HDMEC was observed using an inverted single-photon confocal laser scanning microscope.CD31 expression of HDMEC was detected using flow cytometry.Results Endothelial cells grew after 72 h showing round shape,and most of them were spindle cells on the fifth day.They proliferated rapidly and formed into relatively close fused cells from day 6 to day 10.The center of the colony was round cells,and the surrounding cells were fusiform and radial.In the tenth day,80%cells fused and showed typical morphology of paving stones.Flow cytometry showed that CD31 expression was positive and CD44 expression was negative of HDMEC,whose purity was above 98%.Conclusion The highly purified human dermal microvascular endothelial cells(HDMEC)can be successfully obtained.It is of great significance to the pathogenesis of skin diseases and in-depth study of the biological characteristics and function of HDMEC.

关 键 词：内皮细胞 嘌呤霉素 细胞纯化培养 

分 类 号：R751[医药卫生—皮肤病学与性病学]