干扰素-α诱导HepG2 2.2.15细胞APOBEC3G的表达及其机制

Expression of APOBEC3G induced by interferon-alpha in HepG2 2.2.15 cells

目的探讨干扰素（IFN）-α刺激HepG2 2．2．15细胞后，对载脂蛋白BmRNA编辑酶催化多肽样3G（apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G，APOBEC3G）表达的影响，以及初步探讨Janus激酶-信号传导和转录激活子OAK—STAT）信号通道是否参与APOBEC3G基因转录调控。方法对HepG2 2．2．15细胞给予不同剂量（0、1、10^1、10^2、10^3、10^4 U／mL）IFN-α刺激8h时，以及10^3 U／mL IFN-α刺激2、4、6、8、10、12h时，收集细胞或培养上清液。应用实时荧光定量逆转录聚合酶链反应（RT-PCR）及Western blot检测HepG22．2．15细胞APOBEC3G、STAT-1 mRNA及蛋白的表达水平。应用酶联免疫吸附试验（ELISA）检测细胞培养上清液中乙型肝炎表面抗原与e抗原（HBsAg与HBeAg）水平，应用实时荧光定量PCR及RT-PCR分别检测上清液中HBVDNA水平以及细胞中HBVmRNA水平。结果无IFN-α（0 U／mL）刺激时，HepGa2．2．15细胞APOBEC3G表达水平很低。随着IFN-a浓度的升高，APOBEC3GmRNA及蛋白水平逐步升高，IFN-α浓度为10^4 U／mL时，APOBEC3G表达量最高，并且STAT-1分子mRNA及蛋白的表达量亦逐步升高，与APOBEC3G表达量呈现平行相关。随着IFN-a刺激时间的延长，APOBEC3G表达量明显升高，8h时达到最高，其后逐渐下降。10^4 U／mL IFN-α刺激8h时，HepG2 2．2．15细胞培养上清液中HBsAg、HBeAg、HBVDNA及细胞中HBVmR-NA水平均明显低于无IFN-α刺激的HepG22．2．15细胞。结论WN-a能诱导HepG22．2．15细胞表达APO—BEC3G，在一定范围内，APOBEC3G的表达与IFN—d的剂量、作用时间呈正相关；IFN-α诱导APOBEC3G的表达可能是其发挥抗病毒作用的机制之一；IFN-α是否经JAK-STAT信号通道刺激APOBEC3G的表达，二者之间的关系及其机制尚待进一步研究。

Objective To study the effects of interferon-alpha （IFN-α） on apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G （APOBEC3G） expression by stimulating HepG2 2. 2. 15 cells with IFN-α, and to preliminarily investigate whether Janus kinase-signal transduction and activators of transcription （JAK-STAT） signal pathway participates in the regulation of APOBEC3G gene transcription. Methods HepG2 2. 2. 15 cells were treated with various concentrations of IFN-α（0, 1, 10^1 , 10^2, 10^3, 10^4 U/mL） for 8 hours, or with IFN-α of 10^3 U/mL for 2, 4, 6, 8, 10, 12 hours. In the above-mentioned time, cells or cultural supernatants were collected. The mR NA and protein expression levels of APOBEC3G and STAT-1 in HepG2 2. 2. 15 cells were detected by real-time fluorescent quantitation RT-PCR and Western-blot respectively. The levels of HBsAg and HBeAg in the cultural supernatant of HepG2 2. 2. 15 cells were detected by ELISA. The levels of HBV DNA in supernatant and HBV mRNA in cells were determined by real-time PCR and RT-PCR respectively. Results The expression level of APOBEC3G was very low in HepG2 2. 2. 15 cells untreated with IFN-α （0 U/mL）. With the rising of IFN-α concentration,APOBEC3G mRNA and protein level rose progressively. When IFN-α concentration was 10^4 U/mL, the expression level of APOBEC3G was the highest. Moreover, the expression level of STAT-1 mRNA and protein also rose progressively, which appeared with APOBEC3G expression amount parallelly and relevantly. With the extension of time with IFN-α stimulation, APOBEC3G expression level rose obviously, which reached the highest at the 8 hours, and thereafter dropped gradually. When IFN-α of 10^4 U/mL stimulated 8 hours, the level of HBsAg, HBeAg, HBV DNA in cultural supernatant and the level of HBV mRNA in HepCn 2. 2. 15 cells were obviously lower than the cells untreated with IFN-α. Conclusion IFN-α can induce HepG2 2. 2. 15 cells to express APOBEC3G. Within the certain limits, APOBEC3G expression presents positive correlation with IFN-a dosage and action time. The expression of APOBEC3G induced by IFN-α may be one of antiviral mechanisms of IFN-α. Whether JAK-STAT signal pathway participates in the expression of APOBEC3G induced by IFN-α need further study.