人脐静脉血管内皮细胞体外培养的方研究

人脐静脉血管内皮细胞体外培养的方法研究

蔡维霞1Δ 梁亮2Δ 计鹏1 张万福1 张战凤1 张彦刚1 

肖西峰3 白晓智1 朱华宇1 胡大海1 韩骅2

【摘 要】 目的 建立人脐静脉血管内皮细胞（human umbilical vein endothelial cells，HUVECs）体外高效稳定培养的方法，为组织工程及相关医学基础研究提供稳定的细胞来源。 方法 取自愿捐赠足月妊娠分娩的新生儿脐带，用自制空针软管静脉注入0.1%Ⅱ型胶原酶，置于37℃培养箱中，消化收集，采用含5% FBS及1%内皮细胞生长因子（endothelial cell growth factor,ECGS）的内皮细胞专用培养基进行培养。将消化前后的脐带标本行HE染色，观察HUVECs脱壁情况；流式细胞仪检测原代细胞纯度；细胞培养期间于倒置相差显微镜下观察细胞形态；取第3代HUVECs 行免疫细胞化学染色观察、MTT检测细胞增殖情况，并将细胞接种于细胞外基质胶Matrigel上培养24 h，观察细胞管腔形成情况。 结果 经Ⅱ型胶原酶消化后的脐带内HUVECs大量脱落，细胞消化完全。经Ⅱ型胶原酶37℃培养箱中消化15 min后，可获得纯度为 99.56% 的HUVECs；原代HUVECs培养后2～3 d生长最快，呈典型的铺路石或鹅卵石样排列，4～6 d融合成片。 MTT 法检测显示第3代HUVECs培养后3～4 d细胞生长最快，5 d 左右融合；免疫细胞化学染色显示内皮细胞Ⅷ因子相关抗原表达阳性，培养24 h后在细胞外基质胶Matrigel上可见类似于毛细血管的完整闭合管腔形成。 结论 经自制空针软管静脉注入0.1%Ⅱ型胶原酶，使静脉充分充盈，内皮消化完全，采用含5%FBS和1%ECGS 的内皮细胞专用培养基，可迅速获取大量高纯度、高存活率的HUVECs。

【关键词】 组织工程 种子细胞 人脐静脉血管内皮细胞 细胞培养 

EXPERIMENTAL STUDY ON CULTURE METHOD OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS/CAI Weixia1, LIANG Liang2, JI Peng2, ZHANG Wanfu2, ZHANG Zhanfeng1, ZHANG Yangang1, XIAO Xifeng3, BAI Xiaozhi1, ZHU Huayu1, HU Dahai1, HAN Hua2. 1Burns Center of Chinese PLA, Xijing Hospital, the Fourth Military Medical University, Xi’an Shaanxi, 710032, P.R.China; 2Department of Medical Genetics and Developmental Biology, the Fourth Military Medical University;

3Department of Gynecology and Obstetrics, Tangdu Hospital, the Fourth Military Medical University. Corresponding author: HAN Hua, E-mail: huahan@; HU Dahai, E-mail: burns@

【Abstract】Objective To establish an efficient and stable culture method of human umbilical vein endothelial cells(HUVECs) in vitro so as to provide good source of seed cells for tissue engineered vascular grafts and for precl inical research.Methods The umbilical cords were harvested from full-term normal delivered neonates,which were perfused with

℃

0.1%collagenase II by self-made needle and were digested at37 and5%CO2humidified incubator.The HUVECs were cultured

in endothelial culture medium(ECM)containing5%fetal bovine serum(FBS)and1%endothelial cell growth factor(ECGS).

HE staining of the umbilical cords before and after digestion was used to observe the detachment of HUVECs,flow cytometry to detect the purity of primary HUVECs,and inverted phase contrast microscope to observe the morphology of the cultured HUVECs.The growth of the3rd passage cells was measured by MTT assay;immunocytochemical technique and matrigel-based capillary-like tube formation assay were carried out to identify the function of HUVECs.Results After digestion of0.1%collagenase II,marked HUVECs detachment was observed with complete digestion.The purity of the HUVECs was

℃

99.56%by digestion of0.1%collagenase II at37 and5%CO2humidified incubator for15minutes.Primary HUVECs showed a

cobblestone or pitching stone-like appearance in vitro,forming a confluent monolayer cells after2-3days of culture.MTT assay demonstrated that HUVECs showed the fastest growth speed at3to4days,and showed growth of cell fusion at about5days.

Immunocytochemistry showed that HUVECs highly expressed endothelial marker factor VIII.Matrigel based capillary-like tube formation assay showed that it could form endothelial-like tube structures after24hours of culture.Conclusion Using improved method and ECM could obtain high quantity and high quality primary HUVECs,which might be a kind of promising seed cells for tissue engineering and preclinical research.

作者单位：1 第四军医大学西京医院全军烧伤中心（西安，710032）；2 第四军医大学医学遗传学与发育生物学教研室；3 第四军医大学唐都医院妇产科

△为共同第一作者

通讯作者：韩骅，教授，博士生导师，研究方向：血管发生的分子机制，E-mail: huahan@；胡大海，教授，博士生导师，研究方向：创面愈合与组织修复，E-mail: burns@

网络出版时间：2011-1-6 16:22:41；网络出版地址：/kcms/detail/51.1372.R.20110106.1622.201102.6_001.html