人脐静脉内皮细胞体外分离培养及鉴定.docx

人脐静脉内皮细胞体外分离培养及鉴定【摘要】目的探讨人脐静脉内皮细胞的原代培养方法提高体外分离培养血管内皮细胞的成功率。建立血管内皮细胞培养模型为体外研究血管内皮细胞提供实验基础。方法取2根脐带（至少20cm）冲净淤血采用加工穿刺针固定脐静脉灌注消化液一根用0.2％胶原酶Ⅱ另一根用0.1％胶原酶Ⅰ和0.25％胰酶等比混合消化液比较两种酶的消化效果。收集细胞并用含有10ng/mL的VEGF的培养基中培养观察细胞的生长及传代。并在倒置显微镜下观察细胞的形态学特点同时用免疫组织化学的方法对所得细胞进行鉴定。用流式细胞术观察细胞周期。结果两种消化酶方法均获得了相当数量的人脐静脉内皮细胞胶原酶Ⅱ的消化效果稍优于混合消化酶且比较理想的消化时间均为13min细胞接种后4～5h开始贴壁生长1周左右可生长成单层光镜下呈多角形“铺路石”样排列免疫组织化学法可见内皮细胞胞浆中人第Ⅷ因子相关抗原呈阳性反应。细胞周期显示约有50.6%的细胞处于G0/G1期。结论胶原酶Ⅱ和胶原酶Ⅰ与胰酶等比混合消化液灌注法是获得脐静脉内皮细胞的一种可取方法而胶原酶是分离培养脐静脉内皮细胞的首选消化液成功率高可靠性大可成功构建体外研究血管内皮细胞的模型。【关键词】人脐静脉内皮细胞；细胞培养；流式细胞术；细胞周期；分离；鉴定Cultivationandinvitroidentificationofendothelialcellsfromhumanumbilicalvein〔Abstract〕ObjectiveToexploretheprimaryculturemethodofhumanvascularendothelialcells(ECs)fromumbilicalveinenhancethesuccessrateofseparatingandculturingECsinvitroestablishthemodelofhumanvascularECsandprovideanexperimentalmethodforresearchofECs.MethodsTheumbilicalveinsinhumanumbilicalcords20cmlongwerefixedandfilledthedigestivefluids0.2%collagenenaseⅡinonecordandthecomplexenzymeof0.25%trypsinand0.1%collagenaseⅠinanotherandthedigestiveresultswerecompared.Allthecellswerecollectedandculturedinfluidwith10mg/mLVEGFtheirprocreationandgenerationwereobservedthemorphologiccharacteristicsofECswereobservedwithlightmicroscopyandimmunohistochemistryunderinvertedmicroscopeandthecellcyclewasobservedbyflowcytometer.ResultsEnoughECswereobtainedfrombothdigestiveliquidsandcollagenenaseⅡwasbetterthancomplexenzymetheeffectivedigestingtimewas13minutes.theculturingcellsgrewonthewallappearedafter4-5hoursandthemonolayercellswereformedafteroneweek.ⅧfactorintheECsshowedpositivereactionbyimmunohistochemistry.Cellcyclerevealedthat50.6%cellswereatstageofG0/G1.ConclusionTheperfusionwithcolla〔Keywords〕humanvascularendothelialcellinumbilicalvein;cellculture;flowcytometry;cellcycle;separation;identification血管内皮细胞是位于血管内壁的单层细胞通过产生和分泌许多血管活性物质在维持血管舒缩、抗凝血及血管构建等方面起重要作用同时由于其特殊的解剖学部位使内皮细胞能敏感地感知血流、压力、炎症信号以及血液循环中激素水平的变化通过一系列的信号转导过程与邻近及远处的细胞相互联系对各种刺激作出反应维持机体内外环境的稳定。内皮细胞