用于汉滩病毒固有免疫应答研究的永生化人脐静脉内皮细胞系的建立

Establishment of an immortalized human umbilical vein endothelial cell line used for the study of the innate immune response to Hantaan virus

目的通过导入人端粒酶逆转录酶(hTERT)基因构建永生化人脐静脉血管内皮细胞(HUVEC-hTERT),探索永生化HUVEC对汉滩病毒(HTNV)感染效率及细胞固有免疫调控的影响,评价HUVEC-hTERT作为研究HTNV细胞模型的潜能。方法将hTERT基因克隆入慢病毒载体,构建pCDH-CMV-hTERT-EF1-puro质粒后包装慢病毒并感染HUVEC;嘌呤霉素筛选稳定表达hTERT基因的HUVEC命名为HUVEC-hTERT;利用显微镜观察和免疫荧光细胞化学染色法对HUVEC-hTERT的形态及内皮细胞标志分子人血管性假血友病因子(vWF)、CD31和血管内皮细胞钙黏蛋白(VE-cadherin)进行鉴定;利用免疫荧光法检测HTNV感染后核衣壳蛋白(NP)阳性细胞百分比,观察HTNV对HUVEC和HUVEC-hTERT的感染效率差异;利用实时定量PCR和Western blot法分别检测感染HTNV后,病毒基因组小片段(S)及其编码的NP在不同时间点的复制水平,验证HTNV在细胞中的增殖效率;利用实时定量PCR检测细胞β干扰素(IFN-β)、黏病毒抗性蛋白A(MxA)、MxB、干扰素诱导蛋白2(IFIT2)、干扰素诱导跨膜蛋白3(IFITM3)及炎症因子环加氧酶2(COX2)、细胞间黏附因子(ICAM)和CC趋化因子配体5(CCL5)的mRNA水平并利用Western blot法检测IFIT2、IFITM3和MxA的蛋白水平,观察细胞固有免疫对HTNV的反应是否一致。利用Western blot、实时定量PCR检测感染HTNV后细胞上述分子的表达情况,观察永生化后细胞固有免疫对HTNV的反应是否一致。结果成功筛选出永生化细胞系HUVEC-hTERT,鉴定出其与HUVEC具有相同的表型并表达内皮细胞标志分子vWF、CD31、VE-cadherin;HUVEC-hTERT和HUVEC均可被HTNV感染且感染效率基本相同;在HTNV感染过程中,HUVEC-hTERT的固有免疫分子,如IFN-β、MxA、MxB、IFIT2、IFITM3、COX2、ICAM及CCL5的表达情况与HUVEC相同,表明HUVEC-hTERT固有免疫调控未发生改变。结论HUVEC-hTERT可在一定程度上代替原代HUVEC用于HTNV感染致固有免疫应答调控的研究。

Objective To establish the immortalized human umbilical vein vascular endothelial cells(HUVECs-hTERT)by introducing hTERT gene into primary HUVECs.In order to evaluate the potential of HUVECs-hTERT as a research model of HTNV infection,we explored the infection efficiency of Hantaan virus(HTNV)in HUVECs-hTERT and the influence of celluar innate immune regulation.Methods hTERT gene was cloned into lentivirus vector pCDH-CMV-MCS-EF1-puro,resulting in pCDH-CMV-hTERT-EF1-puro plasmid which was packaged into lentivirus.Then it was infected with HUVECs,and the HUVECs which stably express hTERT gene was selected by using puromycin and named HUVECs-hTERT.The morphology of HUVECs-hTERT and endothelial cell marker molecules,such as human von Willebrand factor(vWF),CD31 and vascular endothelial cell cadherin(VE-cadherin)were identified by microscopic observation and immunofluorescence assay.The percentage of nucleocapsid protein(NP)-positive cells after HTNV infection was detected by immunofluorescence assay to identify thedifference of infection efficiency in HTNV between HUVECs and HUVECs-hTERT.Subsequently,real-time quantitative PCR(RT-qPCR)and Western blot analysis were used to detect the expression of HTNV S mRNA and NP after HTNV infection to verify amplification efficiency of HTNV in HUVECs and HUVECs-hTERT.RT-qPCR were used to detect the mRNA expression level of interferonβ(IFN-β),interferon stimulating gene(ISG),including myxovirus resistance protein A(MxA),myxovirus resistance protein B(MxB),interferon inducing protein 2(IFIT2),interferon-induced transmembrane protein 3(IFITM3)and inflammatory factors,such as cyclooxygenase-2(COX2),intercellular adhesion molecule(ICAM),C-C motif chemokine ligand 5(CCL5)and the protein expression level of IFIT2,IFITM3 and MxA in the two types of cells after HTNV infection to determine whether the cellular innate immune response between HUVECs and HUVECs-hTERT are consistent.Results The immortalized cell line HUVECs-hTERT was screened successfully and the identification results showed that HUVECs-hTERT and HUVECs are with the same phenotype and express endothelial cell marker molecules,such as vWF,CD31 and VE-cadherin.HTNV can infect HUVECs-hTERT and HUVECs with approximately the same efficiency.In HTNV infection,the expression of innate immune molecules,such as IFN-β,MxA,MxB,IFIT2,IFITM3,COX2,ICAM,CCL5 are similar between HUVECs and HUVECs-hTERT,indicating that the innate immune regulation of HUVECs-hTERT has not changed.Conclusion HUVECs-hTERT can replace primary HUVECs for the study of innate immune response regulation during HTNV infection under certain conditions.