逆转录病毒介导的稳定表达E4orf1和绿色荧光蛋白的胎肝窦内皮细胞株的建立

Establishment of Fetal Liver Sinusoidal Endothelial Cells Expressing E4orf1 and GFP by Retroviral System

目的:为构建优化的内皮细胞用于造血干细胞(HSCs)的支持培养,通过逆转录病毒载体系统建立稳定表达腺病毒E4区开放读框1(E4orf1)和绿色荧光蛋白(GFP)的人胎肝窦内皮细胞(HFLSECs)株。方法:利用Plat-A细胞将质粒MSCV-N E4orf1和p MX-GFP分别包装为逆转录病毒,共同感染HFLSECs,将原代培养的HFLSECs和转基因的HFLSECs分别在含有0.5、1、2、4μg/m L嘌呤霉素的EGM-2培养基中培养,以测试最适药物筛选浓度,药筛1周后通过流式细胞分选获得稳定转染E4orf1的GFP^+HFLSECs(E4orf1-GFP/HFLSECs)。通过密度梯度离心法从脐带血中分离单个核细胞,利用免疫磁柱分选获得CD34^+造血干/祖细胞(HSPCs),以E4orf1-GFP/HFLSECs作为饲养层联合SCF、TPO、Flt-3L等3种基础造血相关因子进行体外扩增和半固体造血细胞集落培养。结果:0.5μg/m L嘌呤霉素在5 d内即能完全杀死HFLSECs,而转基因的HFLSECs可以正常存活,以此药物浓度加压筛选1周,后续通过流式分选GFP阳性细胞,阳性率为90.5%,在E4orf1-GFP/HFLSECs饲养层支持下,人脐带血来源的CD34^+细胞15 d扩增了360倍,是单纯细胞因子悬浮培养组的2.2倍,且扩增后的HSPCs在体外仍具有多种造血集落形成能力。结论:该细胞株的建立将为构建造血干细胞体外培养体系及研究造血细胞的发育分化提供适宜的微环境。

Objective ＆ Methods： E4offl, members of 70RFs encoded by early region 4（E4） in adenovirus, can support primary edothelial cells long-term survival in serum-free/growth factor-free conditions by activating Akt and FGF-2/FGF-R1 Pathway. In our study, human fetal liver sinusoid endothelial cells（HFLSECs） expressing E4offl and green fluorescent protein（GFP）（E4offl-GFP/HFLSECs） stably- has been established by retroviral system. The von Willebrand factor（vWF） expression of HFLSECs was identified by immunofluorescence. And the optimal concentration of drug screening was identified based on 0.5, 1, 2, 4 μg/mL puromycin selection. Then E4offl- GFP/HFLSECs was further purified with drug selection and FACS sorting. And the surface marker of E4offl-GFP/ HFLSECs were identified by flow cytometly. Mononuclear cells were isolated fl＇om umbilical cord blood by density gradient centrifugation and CD34＋ hematopoietic stem/progenitor cells（HSPCs） were sorted by anti-CD34 micro- beads. The CD34＋ cells were co-cultured with E4orfl-GFP/HFLSECs in StemSpan medium supplemented with 50 μg/mL SCF, TPO, Flt-3L. Results： GFP＋ HFLSECs transfected with E4orfl was obtained by 0.5 μg/mL puromycin selection and FACS sorting, which maintained normal characteristic in serum-free/proangiogenic factors-free condi- tions. Furthermore, the total nucleated number of CD34＋ cells co-cultured with E4orfl-GFP/HFLSECs increased 360 folds within 15 day-s, which was 2.2 times of the cytokines alone group. More importantly, E4offl-GFP/HFL- SECs maintained colony-forming potential in expanded human umbilical cord blood-derived CD34＋ cells. Conclu- sion： The establishment of E4offl-GFP/HFLSECs will provide a suitable microenvironment not only for ex vivo expansion of HSPCs but also for exploring the development of hematopoiesis.