稳定过表达XAF1基因鼻咽癌放射抗拒CNE-2R细胞株的构建

Optimizing construction of a nasopharyngeal cancer CNE-2R cell stably expressing XAF1

目的构建稳定过表达X连锁凋亡抑制蛋白相关因子1(X-linked inhibitor of apoptosis associated factor 1,XAF1)基因的鼻咽癌放射抗拒CNE-2R细胞株。方法分别将携带XAF1基因感染复数(multiplicity of infection,MOI)为50的慢病毒稀释液、MOI为100的慢病毒原液和MOI为100的慢病毒原液加入终浓度为5μg/m L的聚凝胺(polybrene)转染人鼻咽癌放射抗拒CNE-2R细胞株。通过倒置荧光显微镜观察细胞内荧光数量及强度评判转染效率,通过实时荧光定量逆转录聚合酶链反应(real-time quantitative reversetranscription polymerase chain reaction,q RT-PCR)和蛋白免疫印记法(western blot)检测XAF1基因m RNA和蛋白的表达。结果慢病毒稀释液转染,约60%的细胞可观察到微弱荧光;慢病毒原液转染,约70%的细胞观察到较弱荧光;慢病毒原液加入5μg/m L polybrene的转染效率较高,约90%的细胞观察到较强荧光。嘌呤霉素筛选上述转染效率最高的慢病毒原液加5μg/m L polybrene组细胞,其成功将携带XAF1基因的慢病毒转入鼻咽癌放射抗拒CNE-2R细胞株。q RT-PCR和western blot进一步证实细胞株稳定过表达XAF1基因。结论慢病毒加polybrene转染可成功构建稳定过表达XAF1基因的鼻咽癌放射抗拒CNE-2R细胞株。

Objective To examine methods for constructing a nasopharyngeal cancer cell line（CNE-2R） stably expressing （X-linked inhibitor of apoptosis associated factor 1）XAF1. Method CNE-2R cells were infected with lentivirus encoding XAF1 as well as a green fluorescent protein reporter. Cells were infected at multiplicities of infection（MOI） of 50 or 100 without polybrene, or at an MOI of 100 in the presence of polybrene（5 μg/mL）. Expression of XAF1 mRNA and protein was verified using quantitative RT-PCR and Western blotting. Results Faint fluorescence was detected in 60% of CNE-2R cells after transfection with lentivirus at an MOI of 50. A slightly higher percentage（70%） of CNE-2R cells showed weak fluorescence after transfection with lentivirus at an MOI of 100. When cells were infected at an MOI of 100 in the presence of polybrene, 90% of CNE-2R cells showed strong fluorescence. Stably transfected cell lines were selected using puromycin. Conclusion The efficiency with which XAF1-expressing lentivirus can infect CNE-2R cells is closely related to the state of the cells, MOI, and polybrene concentration. Addition of appropriate concentrations of polybrene to lentivirus can increase efficiency of infection. Stably transfected cell lines can be selected with puromycin.