人脐静脉血管内皮细胞体外培养的方法研究

EXPERIMENTAL STUDY ON CULTURE METHOD OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS

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。

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 preclinical 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 at 37 and 5% CO2 humidified incubator.The HUVECs were cultured in endothelial culture medium（ECM） containing 5% fetal bovine serum（FBS） and 1% 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 the 3rd 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 of 0.1% collagenase II,marked HUVECs detachment was observed with complete digestion.The purity of the HUVECs was 99.56% by digestion of 0.1% collagenase II at 37 and 5% CO2 humidified incubator for 15 minutes.Primary HUVECs showed a cobblestone or pitching stone-like appearance in vitro,forming a confluent monolayer cells after 2-3 days of culture.MTT assay demonstrated that HUVECs showed the fastest growth speed at 3 to 4 days,and showed growth of cell fusion at about 5 days.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 after 24 hours 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.