微血管内皮细胞对小鼠造血干细胞增殖的影响  

The effect of microvascular endothelial cells on the proliferation of hematopoietic stem cells in mice

作　　者：钱怡 杨敏 林凡莉 汪姝玥 李晓明 黄纯兰 QIAN Yi;YANG Min;LIN Fanli;WANG Shuyue;LI Xiaoming;HUANG Chunlan(Department of Hematology,the Affiliated Hospital of Southwest MedicalUniversity,Luzhou,Sichuan 646000,Chin)

机构地区：[1]西南医科大学附属医院血液内科,四川泸州646000

出　　处：《重庆医学》2018年第18期2408-2412,2417,共6页Chongqing medicine

基　　金：国家自然基金资助项目(81450030)

摘　　要：目的探索微血管内皮细胞(MECs)对造血干细胞(HSCs)增殖的影响,以建立一种促进HSCs增殖的方法。方法选取C57BL/6小鼠和C57系GFP小鼠,处死获取肺叶组织。(1)MECs的分离、培养和鉴定:肺组织经Ⅰ型胶原酶消化获得的细胞在含20%胎牛血清(FBS)、2ng/mL血管内皮生长因子(VEGF)、100U/mL肝素、0.1mg/mL青霉素及链霉素的DMEM-F12培养基培养,第8天进行CD31荧光鉴定,"铺路石"细胞用于共培养并以流式细胞仪(FCM)检测八因子相关抗原(vWF)、CD31、CD34、CD45表达率。(2)GFP小鼠HSCs的分离、鉴定:密度梯度离心法和MACs-CD117+磁珠法分选HSCs,FCM检测纯度及CD117CD34共表达率。(3)MECs促HSCs增殖:设立对照组(HSCs)、共培养组(MECs+HSCs),培养7d,观察HSCs生长情况并计数、计算扩增倍数;第7天,收集共培养组HSCs,FCM检测CD117CD34共表达率。结果(1)肺MECs:第3天形成细胞簇,第6天成"血管样"改变,第14天为"铺路石"外观。培养第8天CD31阳性率为54.5%。vWF、CD31、CD34、CD45阳性率分别为81.39%、45.80%、57.48%、0.17%。(2)平均每只小鼠骨髓经MACs分选后可获约4.7×105个CD117+细胞,纯度为99.51%。(3)随着培养时间的延长,两组HSCs计数均有所增加,但共培养组HSCs扩增倍数大于对照组(P<0.05)。第1~7天共培养组与对照组细胞数相对变化倍数分别为1.21、1.35、1.50、1.72、1.71、1.75和1.78(P<0.05),第4天变化最明显。共培养7d后HSCs的CD117CD34共表达率为92.06%。结论 MECs对HSCs增殖有促进作用,共培养第4天促进作用最明显,并且MECs可以促进HSCs CD34的表达。Objective To explore the effect of microvascular endothelial cells（MECs）on the proliferation of hematopoietic stem cells（HSCs）,in order to establish a method to promote the proliferation of HSCs.Methods The C57 BL/6 and C57 GFP mice were selected and sacrificed to obtain lung tissues.（1）Isolation,culture and identification of MECs：MECs were obtained by digestion of lung tissue with type Ⅰcollagenase and cultured in DMEM-F12 supplemented with 20% FBS,2 ng/mL VEGF and 100 U/mL heparin,and were identified by immunofluorescence assay with FITC-CD31 at the 8 th day after isolation.The expression rates of vWF,CD31,CD34 and CD45 of the cobblestone-like cells were detected by flow cytometry（FCM）,and the cells were used for co-culture in the subsequent experiments.（2）Isolation and identification of GFP mice HSCs：density gradient centrifugation and MACs-CD117＋magnetic beads were performed to isolate HSCs,and the purity and the co-expression of CD117 and CD34 were detected by FCM.（3）Investigation of the promoting effects of MECs on the HSCs proliferation：all cells in the control group（HSCs）and the co-culture group（MECs＋HSCs）were cultured for 7 days,then observed the status of HSCs,counted and calculated the relative fold changes.At the 7 th day,the expression ratio of CD117＋CD34＋HSCs in the co-culture group were detected by FCM analysis.Results（1）Pulmonary MECs：the cell clusters formed at the 3 th day,the vascular like changes emerged at the 6 th day,apaving stone appearance formatted at the 14 th day.The positive rate of CD31 was 54.5% at the 8 th day.The positive rates of vWF,CD31,CD34 and CD45 were 81.39%,45.80%,57.48% and 0.17% respectively.（2）On average,4.7×10^5 CD117^＋ cells were obtained from each mice bone marrow,and the purity was about 99.51%.（3）The number of HSCs were increased in the two groups,accompanied by the extension of cultivation time.While the co-culture group increased significantly than the control group（P〈0.05）.The ratios（relati

关 键 词：微血管内皮细胞 造血干细胞 增殖 

分 类 号：R551.3[医药卫生—血液循环系统疾病]