人脐静脉内皮细胞的分离培养及条件复制性腺病毒Adv5/dE1A-Aschk1对该细胞的杀伤作用

背景与目的:复制性腺病毒因其高效转导、大量复制等优点而成为目前基因治疗领域最为重要的载体之一,腺病毒体内应用时对血管内皮的作用是评估其安全性和副作用的重要指标.本研究探讨条件复制性腺病毒对增生期和静止期的血管内皮细胞的生长影响.方法:脐静脉内灌注消化液分离内皮细胞,所获内皮细胞体外培养,经免疫荧光染色及扫描电镜检查,鉴定所获内皮细胞及其纯度;细胞病变实验比较条件复制性腺病毒Adv5/dE1A-Aschk1对人宫颈癌细胞株HeLa细胞和血管内皮细胞的裂解效应;四甲基偶氮唑盐(MTT)法检测不同滴度腺病毒对血管内皮细胞的增殖抑制作用;流式细胞仪分析腺病毒单用或联合放射线作用对细胞凋亡的影响.结果:血管内灌注消化液法可获取高纯度的内皮细胞;与HeLa细胞相比,血管内皮细胞对条件复制性腺病毒Adv5/dE1A-Aschk1较耐受,500 MOI的病毒滴度感染静止态内皮细胞未出现明显的裂解效应;Adv5/dE1A-Aschk1对内皮细胞的增殖抑制效应随病毒滴度的增加而增强,100 MOI的病毒作用96 h,增殖态内皮细胞增殖抑制率为(22.0±2.5)%,而静止态内皮细胞为(13.0±2.3)%;Adv5/dE1A-Aschk1感染联合放射线处理时,增殖态内皮细胞较静止态更为敏感,两者凋亡细胞比例分别为(35.7±3.0)%、(23.1±2.5%)%(P＜0.05).结论:体外分离的人脐静脉内皮细胞可作为探讨条件复制性腺病毒效应的模型细胞;条件复制性腺病毒Adv5/dE1A-Aschk1损伤血管内皮细胞的病毒剂量高于杀伤肿瘤细胞的有效剂量,有良好的治疗窗;增殖态血管内皮与静止态相比,更易被复制性腺病毒杀伤.

Effects of conditionally replicating adenovirus Adv5/dE1A-Aschk1 on isolated human umbilical endothelial cells

BACKGROUND & OBJECTIVE: Replicating adenovirus, with the favorable features of efficient transduction and multiple in malignant cells, represent a promising option for gene therapy. Its influence on vascular endothelia in vivo is an important indicator for safety evaluation. This study was to explore the effects of conditionally replicating adenovirus Adv5/dE1A-Aschk1 on proliferating and resting endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVECs) were isolated by filling umbilical veins with digestive enzyme solution. HUVECs were cultured and observed with immunofluorescent staining and under electron microscope to identify endothelial cells. The oncolytic effects of Adv5/dE1A-Aschk1 on HeLa and endothelial cells were measured by cytopathic effect assay (CPE)? its effect on the proliferation of endothelial cells was measured by MTT assay. Cell apoptosis after treatment of Adv5/dE1A-Aschk1 alone or in combination with irradiation was measured by flow cytometry (FCM). RESULTS: Highly purified endothelial cells were isolated. Adv5/dE1A-Aschk1 lysed HeLa cells in a replication-dependent fashion, but did not exhibit detectable cytopathic effects on resting endothelial cells at a multiplicity of infection (MOI) of 500. The inhibitory effect of Adv5/dE1A-Aschk1 on the proliferation of endothelial cells increased with the increase of viral titer. When treated with Adv5/dE1A-Aschk1 at a MOI of 100 for 96 h, the proliferation inhibition rate of proliferating HUVECs was (22.0+/-2.5)%, but that of resting HUVECs was (13.0+/-2.3)%. When treated with Adv5/dE1A-Aschk1 combined irradiation, the apoptosis rate of resting HUVECs was (23.1+/-2.5)%, and that of proliferating HUVECs was (35.7+/-3.0)%. CONCLUSIONS: HUVECs may be taken as a good target for the research of conditionally replicating adenovirus. The viral titer for injuring endothelial cells is much higher than that for killing tumor cells, so Adv5/dE1A-Aschk1 has a wide range of therapeutic dosage. As compared with resting HUVECs, proliferating HUVECs are more likely to be lysed by Adv5/dE1A-Aschk1.