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目的:探讨在肺泡上皮细胞模型中甲型H1N1流感病毒（H1N1病毒）诱导低氧诱导因子-1α（HIF-1α）核转位的分子机制。方法:体外培养人肺腺癌上皮细胞（A549细胞），选取对数生长期细胞用于实验。①实验1：采用感染复数（MOI）1.0的H1N1病毒感染细胞24 h，建立H1N1病毒感染的A549细胞模型（H1N1病毒感染组）；并设立空白对照组。采用蛋白质免疫印迹试验（Western Blot）检测细胞中核转运受体蛋白（Importin 4、Importin 7）表达，探讨HIF-1α核转位是否依赖Importin 4、Importin 7。②实验2：采用不同MOI（0、0.1、0.5、1.0、2.0、4.0）的H1N1病毒感染细胞24 h；采用MOI 1.0的H1N1病毒感染细胞0、3、6、12、18、24、36 h。采用实时荧光定量反转录-聚合酶链反应（RT-PCR）检测细胞中胞裂蛋白9基因变异剪接体1（SEPT9_i1）mRNA表达，探讨不同MOI和感染时间对SEPT9_i1表达的影响。③实验3：采用小干扰RNA（siRNA）转染细胞24 h抑制SEPT9_i1，建立沉默SEPT9_i1的A549细胞模型（siRNA-SEPT9_i1组）；并设立空白对照组和空白载体对照组（siControl组）。3组细胞转染24 h后均给予MOI 1.0的H1N1病毒感染24 h，采用实时荧光定量RT-PCR检测细胞中SEPT9_i1 mRNA表达，以探讨siRNA对SEPT9_i1的干扰效率。④实验4：将细胞分为siControl组和siRNA-SEPT9_i1组，两组细胞转染方法同实验3。转染24 h后，两组均以MOI 1.0的H1N1病毒感染细胞，于24 h采用免疫荧光法观察细胞中HIF-1α核内外分布情况；于6、12、24、36、48 h采用实时荧光定量RT-PCR检测病毒M基因表达，以明确SEPT9_i1对HIF-1α核转位和病毒复制的影响。⑤实验5：将细胞分为空白对照组（完全培养基）、SP600125组〔100 μmol/L的c-Jun氨基末端激酶（JNK）信号通路抑制剂SP600125处理细胞2 h〕、H1N1病毒感染组（MOI 1.0的H1N1病毒感染细胞24 h）和H1N1病毒+ SP600125组（100 μmol/L的SP600125预处理2 h后，再加入MOI 1.0的H1N1病毒感染细胞24 h）。采用实时荧光定量RT-PCR检测细胞中SEPT9_i1 mRNA和病毒M基因表达，探讨JNK信号通路对SEPT9_i1表达及病毒复制的影响。结果:①实验1：与空白对照组相比，H1N1病毒感染组A549细胞中Importin 4和Importin 7蛋白表达差异均无统计学意义〔Importin 4蛋白（Importin 4/GAPDH）：1.08±0.03比1.05±0.03，Importin 7蛋白（Importin 7/GAPDH）：0.87±0.11比0.78±0.03，均n P>0.05〕。说明H1N1病毒感染A549细胞中HIF-1α核转位可能不依赖Importin 4和Importin 7。②实验2：A549细胞中SEPT9_i1 mRNA表达随H1N1病毒MOI增加及感染时间延长呈升高趋势，分别于MOI 2.0和感染18 h达峰值，与MOI为0或感染0 h比较差异均有统计学意义（2n -ΔΔCT：MOI 2.0为1.39±0.05比1.00±0.00，18 h为1.47±0.04比1.00±0.00，均n P<0.01）。说明H1N1病毒感染A549细胞中SEPT9_i1表达与病毒MOI及感染时间相关。③实验3：与空白对照组比较，siRNA-SEPT9_i1组A549细胞中SEPT9_i1 mRNA表达明显下调（2n -ΔΔCT：0.38±0.11比1.00±0.00，n P0.05）。说明沉默SEPT9_i1可抑制H1N1病毒感染A549细胞中SEPT9_i1表达。④实验4：siRNA-SEPT9_i1组H1N1病毒感染A549细胞中HIF-1α核转位较siControl组明显减少。siControl组感染H1N1病毒后细胞中病毒M基因表达逐渐升高，48 h达峰值；siRNA-SEPT9_i1组A549细胞中病毒M基因表达明显下调，48 h与siControl组比较差异有统计学意义（2n -ΔΔCT：3.47±0.66比8.17±0.38，n P<0.05）。说明沉默SEPT9_i1后可以抑制H1N1病毒感染A549细胞中HIF-1α核转位和病毒复制。⑤实验5：H1N1病毒感染组A549细胞中SEPT9_i1 mRNA和病毒M基因表达较空白对照组明显升高；而H1N1病毒+ SP600125组A549细胞中SEPT9_i1 mRNA和病毒M基因表达均明显低于H1N1病毒感染组（2n -ΔΔCT：SEPT9_i1 mRNA为0.12±0.10比1.53±0.14，病毒M基因为2.13±0.10比4.66±0.14，均n P 0.05]. These indicated that the HIF-1α nuclear translocation in A549 cells might not be independent of Importin 4 and Importin 7 during H1N1 virus infection. ② Experiment 2: the SEPT9_i1 mRNA expression in A549 cells was increased with the increase in MOI and infection time of H1N1 virus, and peaked at MOI 2.0 or 18 hours after infection, and the differences were statistically significant as compared with MOI 0 or 0 hour after infection (2 n -ΔΔCT: 1.39±0.05 vs. 1.00±0.00 at MOI 2.0, 1.47±0.04 vs. 1.00±0.00 at 18 hours, both n P < 0.01). This indicated that the SEPT9_i1 expression in A549 cells was related to the MOI and the infection time during H1N1 virus infection. ③ Experiment 3: compared with the blank control group, the SEPT9_i1 mRNA expression in A549 cells was significantly decreased in the siRNA-SEPT9_i1 group (2 n -ΔΔCT: 0.38±0.11 vs. 1.00±0.00, n P 0.05). This indicated that SEPT9_i1 silence could inhibit the expression of SEPT9_i1 mRNA in H1N1 virus-infected A549 cells. ④ Experiment 4: HIF-1α nuclear translocation in the H1N1 virus-infected A549 cells in the siRNA-SEPT9_i1 group was significantly reduced as compared with the siControl group. The virus M gene expression after H1N1 virus infection in the siControl group was gradually increased, and peaked at 48 hours. The expression of virus M gene in A549 cells in the siRNA-SEPT9_i1 group was significantly down-regulated, and showed a statistically significant difference at 48 hours as compared with the siControl group (2 n -ΔΔCT: 3.47±0.66 vs. 8.17±0.38, n P < 0.05). This indicated that HIF-1α nuclear translocation and virus replication in H1N1 virus-infected A549 cells were inhibited after silencing SEPT9_i1. ⑤ Experiment 5: the expressions of SEPT9_i1 mRNA and virus M gene in A549 cells in the H1N1 virus infection group were significantly higher than those in the blank control group. However, the expressions of SEPT9_i1 mRNA and viral M gene in A549 cells in the H1N1 virus+SP600125 group were significantly lower than those in the H1N1 virus infection group (2 n -ΔΔCT: SEPT9_i1 mRNA was 0.12±0.10 vs. 1.53±0.14, viral M gene was 2.13±0.10 vs. 4.66±0.14, both n P < 0.05). There was no significant difference in above indicators between the SP600125 group and the blank control group. This indicated that the JNK signaling pathway could regulate the expression of SEPT9_i1 in A549 cells during H1N1 virus infection, and the JNK signaling pathway inhibition could down-regulate the expression of SEPT9_i1 and inhibit virus replication.n Conclusion:The H1N1 virus regulates the expression of SEPT9_i1 by activating the JNK signaling pathway, thus increase HIF-1α transport efficiency and H1N1 replication.