Seo Young-Jun, Kim Gi-Hyun, Kwak Ho-Jung, Nam Ju-Sun, Lee Hwa Jeong, Suh Soo-Kyung, Baek Kyung-Min, Sohn Yeo-Won, Hong Seung-Hwa
Advanced Therapy Products Research Division, National Institute of Food and Drug Safety Evaluation, Korea Food and Drug Administration, Seoul 122-704, Republic of Korea.
Pharmacology. 2009;84(3):135-44. doi: 10.1159/000235158. Epub 2009 Aug 14.
Although antiviral assays have been the most widely available biological assays for interferons (IFNs), they are less sensitive and provide considerable interassay variation. In this study, we demonstrate a new reporter cell line, which is based on HeLa cells transfected with a plasmid containing a human Mx2 promoter driving a luciferase (Luc) cDNA. To characterize the specific gene expression profiles induced by interferon alpha, we analyzed the microarray results of interferon response gene expression induced by IFN-alpha2a or IFN-alpha2b treatment with HeLa cells. We found that the Mx2 gene increased the most by treatment with both IFN-alpha2a and IFN-alpha2b. Based on this result, we designed a reporter cell line, HeLa-Mx2, suitable for determination of IFN-alpha. HeLa cells were stably transfected with the luciferase gene under the control of Mx2 promoter. The expression of luciferase can be easily measured for luminescence using a 96-well luminometer and has been correlated with the concentration of added IFN and cell density. In the validation results, our reporter cell line had specificity for type I IFN, but the significant effects of a number of other cytokines such as tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-2, IL-5, IL-6 and GM-CSF, or type II interferon (IFN-gamma) were not observed. Moreover, the robustness of our cell line is demonstrated by the lack of an effect of the HeLa-Mx2 cell culture's age on the performance of the reporter gene assay. The reporter gene assay demonstrated reproducible dose-response curves for IFN-alpha2a in the range of 1-10,000 IU/ml. The 95% confidential limit and total coefficient of variation estimates ranged between 96 and 116 and 10.51% in the reducible range mentioned above, respectively. In conclusion, we established a stable IFN-responsible HeLa-Mx2 cell line, which has advantages with regard to simplicity, selectivity, and reliability over conventional cytopathic effect reduction assays used to quantify IFN-alpha activity.
尽管抗病毒检测一直是干扰素（IFN）最广泛使用的生物学检测方法，但它们的敏感性较低，且检测间差异较大。在本研究中，我们展示了一种新的报告细胞系，它基于用含有驱动荧光素酶（Luc）cDNA的人Mx2启动子的质粒转染的HeLa细胞。为了表征干扰素α诱导的特定基因表达谱，我们分析了用IFN-α2a或IFN-α2b处理HeLa细胞后干扰素反应基因表达的微阵列结果。我们发现，用IFN-α2a和IFN-α2b处理后，Mx2基因的增加最为显著。基于这一结果，我们设计了一种适用于测定IFN-α的报告细胞系HeLa-Mx2。HeLa细胞用在Mx2启动子控制下的荧光素酶基因进行稳定转染。使用96孔发光计可以很容易地测量荧光素酶的表达以检测发光，并且其与添加的IFN浓度和细胞密度相关。在验证结果中，我们的报告细胞系对I型干扰素具有特异性，但未观察到许多其他细胞因子如肿瘤坏死因子-α、白细胞介素（IL）-1β、IL-2、IL-5、IL-6和粒细胞-巨噬细胞集落刺激因子（GM-CSF）或II型干扰素（IFN-γ）的显著影响。此外，HeLa-Mx2细胞培养物的年龄对报告基因检测性能没有影响，这证明了我们细胞系的稳健性。报告基因检测在1-10,000 IU/ml范围内显示了IFN-α2a可重复的剂量反应曲线。在上述可降低范围内，95%置信限和总变异系数估计值分别在96至116和10.51%之间。总之，我们建立了一种稳定的对IFN有反应的HeLa-Mx2细胞系，与用于量化IFN-α活性的传统细胞病变效应降低检测相比，它在简单性、选择性和可靠性方面具有优势。
