摘要
目的构建人血管紧张素转化酶2(ACE2)稳定表达的人永生化二型肺泡上皮细胞(HPAEpiC)和支气管上皮细胞(Beas-2b),并探索其作为穿山甲冠状病毒GX_P2V感染模型的应用。方法本研究为实验研究。根据ACE2基因序列设计合成特异性引物并通过提取人克隆结肠癌细胞(Caco-2)的基因组作为模板对目的基因进行扩增,将扩增后的目的基因定向连接至经限制性内切酶BmTI/AbsI酶切后的pCDH-CMV载体上,构建pCDH-CMV-ACE2重组质粒。经筛选和测序无误后,将阳性克隆转染人永生化胚胎肾细胞(HEK-293T)包装慢病毒。将慢病毒转染HPAEpiC和Beas-2b细胞,分为稳转细胞株(HPAEpiC-ACE2组、Beas-2b-ACE2组)和野生细胞株(HPAEpiC-WT组、Beas-2b-WT组)。使用凝胶电泳、逆转录聚合酶链反应(RT-PCR)和蛋白质印迹法技术验证ACE2的稳定表达。CCK8法对比稳转细胞株和野生细胞株的增殖能力。利用稳定表达ACE2的2种细胞株(HPAEpiC-ACE2组、Beas-2b-ACE2组)测试GX_P2V的感染情况。结果重组质粒pCDH-CMV-ACE2在慢病毒的介导下转染至HPAEpiC和Beas-2b细胞。RT-PCR结果显示,HPAEpiC-ACE2组ACE2 mRNA表达较HPAEpiC-WT组升高[(23.749±0.557)比(1.002±0.088)],Beas-2b-ACE2组ACE2 mRNA表达较Beas-2b-WT组升高[(12.988±0.330)比(1.000±0.052)],差异均有统计学意义(t值分别为69.75、48.45,均P<0.001),表达效率均提高了10倍以上。蛋白质印迹法结果显示,HPAEpiC-ACE2组和Beas-2b-ACE2组ACE2蛋白均稳定表达,而HPAEpiC-WT组和Beas-2b-WT组均无ACE2蛋白的表达。CCK8法检测结果显示,HPAEpiC-WT组与HPAEpiC-ACE2组[(0.323±0.002)比(0.311±0.017)]、Beas-2b-WT组与Beas-2b-ACE2组细胞增殖能力[(0.325±0.030)比(0.312±0.005)]比较差异均无统计学意义(t值分别为1.24、0.68,均P>0.05)。RT-PCR结果显示,随着时间延长,HPAEpiC-WT组和Beas-2b-WT组相对病毒RNA含量近乎成一条直线,表明HPAEpiC-WT和Beas-2b-WT对GX_P2V均不易感;而HPAEpiC-ACE2组和Beas-2b-ACE2组相对病毒RNA含量呈现明显的增加趋势,且病毒RNA含量在HPAEpiC-ACE2组和Beas-2b-ACE2组中约是野生细胞株的1580倍、10500倍。结论成功构建了稳定表达ACE2的HPAEpiC和Beas-2b细胞,相较于野生细胞株,稳转细胞株可显著感染GX_P2V,为呼吸道病原体的研究提供了人源肺部非肿瘤细胞系的感染模型。
ObjectiveTo construct immortalized human typeⅡalveolar epithelial cell line(HPAEpiC)and bronchial epithelial cell line(Beas-2b)stably expressing human angiotensin-converting enzyme 2(ACE2),and to explore their application as an infection model for the pangolin coronavirus GX_P2V.MethodsThis was an experimental study.Specific primers were designed and synthesized based on the ACE2 gene sequence.The target gene was amplified using genomic DNA extracted from Caco-2 cells as a template.The amplified target gene was directionally ligated into the pCDH-CMV vector that was digested with the restriction enzymes BmTI and AbsI,to construct the pCDH-CMV-ACE2 recombinant plasmid.After screening and sequencing verification,the positive clones were transfected into HEK-293T cells for lentivirus packaging.HPAEpiC and Beas-2b cells were transfected with the lentivirus,thus constructing the stable cell lines(HPAEpiC-ACE2 group and Beas-2b-ACE2 group)and wild-type cell lines(HPAEpiC-WT group and Beas-2b-WT group).Gel electrophoresis,reverse transcription-polymerase chain reaction(RT-PCR),and Western blotting were used to verify the stable expression of ACE2.The proliferation capacity of the stable and wild-type cell lines was compared using the CCK-8 assay.The susceptibility of the two stable ACE2-expressing cell lines(HPAEpiC-ACE2 and Beas-2b-ACE2)to GX_P2V infection was also tested.ResultsThe recombinant plasmid pCDH-CMV-ACE2 was successfully transfected into HPAEpiC and Beas-2b cells.RT-PCR results showed that the mRNA expression of ACE2 in the HPAEpiC-ACE2 group was significantly higher than the HPAEpiC-WT group(23.749±0.557 vs 1.002±0.088,t=69.75,P<0.001),and it was significantly higher in the Beas-2b-ACE2 group than the Beas-2b-WT group(12.988±0.330 vs 1.000±0.052,t=48.45,P<0.001),indicating more than 10-fold increase in expression efficiency.Western blotting confirmed stable protein expression of ACE2 in the HPAEpiC-ACE2 and Beas-2b-ACE2 cells,which was not detected in the HPAEpiC-WT and Beas-2b-WT cells.The CCK-8 assay showed no significant differences in the proliferation capacity between the HPAEpiC-WT and HPAEpiC-ACE2 groups(0.323±0.002 vs 0.311±0.017,t=1.24,P>0.05),and between the Beas-2b-WT and Beas-2b-ACE2 groups(0.325±0.030 vs 0.312±0.005,t=0.68,P>0.05).RT-PCR results indicated that the relative viral RNA content in the HPAEpiC-WT and Beas-2b-WT groups remained nearly constant over time,suggesting that these wild-type cell lines were not susceptible to GX_P2V infection.In contrast,the relative viral RNA content in the HPAEpiC-ACE2 and Beas-2b-ACE2 groups showed a significant increasing trend,with viral RNA levels approximately 1580-fold and 10500-fold higher than the wild-type cell lines,respectively.ConclusionHPAEpiC and Beas-2b cells stably expressing ACE2 are successfully constructed.Compared with wild-type cell lines,the stable cell lines exhibit a significant susceptibility to GX_P2V infection,providing a human non-tumor lung cell-based infection model for studying respiratory pathogens.
作者
余忠阔
曹彦
顾少岩
解立新
Yu Zhongkuo;Cao Yan;Gu Shaoyan;Xie Lixin(College of Pulmonary and Critical Care Medicine,Chinese PLA General Hospital,Beijing 100091,China)
出处
《国际呼吸杂志》
2025年第5期382-389,共8页
International Journal of Respiration
基金
国家自然科学基金(82172109)。
