猪脐静脉血管内皮细胞的分离及体外培养研究

为建立一种操作简单并且能获得大量猪血管内皮细胞的分离培养方法,本研究采集新生健康长白猪脐带,取其脐静脉血管,灌注0.1%的Ⅰ型胶原酶,于37℃水浴消化10 min后收集内皮细胞,培养于含20%胎牛血清的M199培养基中,长满单层后用胰酶消化传代培养。并对培养的细胞形态学、相关抗原和其他特征以及对病毒的易感性进行鉴定。结果表明,Ⅰ型胶原酶消化后可获得大量内皮细胞,细胞培养后的形态学观察显示细胞贴壁生长良好,呈典型的铺路石状排列;第Ⅷ因子相关抗原和细胞摄取乙酰化低密度脂蛋白均呈阳性,内皮细胞比率可高达100%。传3代后的生长曲线显示细胞传代后延迟期小于1 d,对数生长期约3 d,第5 d进入平台期,第7 d开始脱落死亡。病毒感染试验表明所获得的内皮细胞对猪瘟病毒高度易感,病毒生长与常用的PK-15没有区别。上述研究结果表明本研究建立的血管内皮细胞分离培养方法简便有效,为大量制备原代血管内皮细胞提供了可靠的方法。

Isolation and culture of swine umbilical vein endothelial cells

To establish a simple method to isolate and cultivate swine umbilical vein endothelial cell(SUVEC),vein vessels were separated from umbilical cords of newborn piglets and infused with 0.1% collagenase I solution,incubating at 37 ℃ for 10 min.Endothelial cells(ECs) were flushed out and cultured in M199 medium supplemented with 20% FBS.The confluence ECs were subcultured by trypsin digestion and characterized.Results showed that abundant ECs could be obtained with collagenase digestion followed by flush and grew as a confluent monolayer with cobblestone morphology,showing positive for both factor VIII-related antigen and Dil-acetylated low density lipoprotein uptake.The percentage of ECs in the culture were reached up to 100%,with growth curve showing lag phase,the log phase,the plateau phase after passage at about 1,3 and 5 days,respectively.In addition,the cultured ECs were also as susceptible to the infection of swine fever virus as routine PK-15 cells.These results indicated the method established in this experiment was simple and effective to prepare SUVEC.