| Objective: To study the expression of the chemonkine SDF1 in the ATⅡAECs(A549 cells) in normal and hypoxia condition,also to clarify whether SDF1 is regulated by HIF1,or HIF2 or the both.Methods: A549 cells were cultured under hypoxic (1.5%O2 concentration )or normal conditions for 48 hours ,and then realtime RT-PCR was used to determine the mRNA expression of SDF1, HIF1αand HIF2αin the two groups.Next,the siHIF1α,siHIF2αand scrambled control also negative control were transferred into the A549 cells with lip2000 for 48 hours all under hypoxic condition .Also the mRNA expresssion for three genes above was qaulitified by realtime RT-PCR in all the groups. Whole cell extracts were separated by 10% SDS-PAGE and transferred to PVDF membrane. Immunoblotting was performed using HIF-1α, HIF-2αand beta-actin antibodies.At the same, SDF1 protein levels in culture media were measured using a commercial elisa kit.Results: The mRNA expression of HIF1αand HIF2αcould be detected and the SDF1 was almost misssing under normal condition ;in contrast,the levels of the three were markedly upregualted under the hypoxic contidion for 48 hours(p﹤0.05). The mRNA levels of HIF1αand SDF1 were decreased in the siHIF1αgroup and the levels of HIF2αand SDF1 declined in the siHIF1αgroup in contrast with the scrambled control .And the difference has statistical significance.It was demonstrated that the siRNA sequences were effective.Conclusion: Hypoxia could upregulate SDF1 in the Human lung adenocrcinoma cells A549 derived from ATⅡAECs and may be mediated by HIF1 and HIF2,in which there was a cooperation between the two transcription factors. This finding will be an aid to claryfy the mechanism why SDF1 is strongly upregulated in reactive hyperplastic alveolar epithelial cells and how SDF1 recruits fibrocytes from the blood participating in the fibrogenesis of IPF. Also this may be a new evidence to support the pivotal role of HIFs/SDF1 in the pathogenesis of IPF and the significance as therapeutic targets. |
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