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目的研究Netrin-1对肺泡上皮细胞钠离子通道α亚基(epithelial sodium channelα-subunit,α-ENaC)表达的调控及其可能的机制,并探讨其在急性肺损伤(acute lung injury,ALI)及急性呼吸窘迫综合征(acute respiratory distresssyndrome,ARDS)中的作用。方法取对数生长期的A549细胞,用相同浓度的Netrin-1(500μg/L),分别作用0、3、6、12、24和48 h,用不同浓度的Netrin-1(0、0.5、5、50和500μg/L),作用12 h。将A549细胞分为药物组(500μg/LNetrin-1)、抑制组(1μmol/L PSB1115+500μg/L Netrin-1)和对照组(仅加RPMI1640培养基),培养12 h。RT-PCR法分别检测各组α-ENaC基因mRNA的转录水平;Western blot法检测各组α-ENaC蛋白的表达水平。结果相同浓度下,Netrin-1作用A549细胞6 h后,α-ENaC基因mRNA相对转录水平及蛋白相对表达量显著高于对照组(P<0.05),作用12 h时达最高;相同时间内,Netrin-1浓度大于5μg/L时,α-ENaC基因mRNA相对转录水平及蛋白的相对表达量均明显高于对照组(P<0.05),且呈剂量依赖性,浓度为500μg/L时达最高;药物组α-ENaC基因mRNA相对转录水平及蛋白相对表达量均明显高于对照组及抑制组(P<0.05),PSB1115可明显抑制α-ENaC基因mRNA的转录和蛋白表达。结论 Netrin-1可通过腺苷A2b受体途径,从基因水平上调α-ENaC的表达,有益于ALI及ARDS的预后。
Objective To investigate the regulation of Netrin-1 on the expression of α-subunit (α-ENaC) in alveolar epithelial cells and to explore the possible mechanism of Netrin-1 in the pathogenesis of acute lung injury (ALI) and Acute respiratory distress syndrome (acute respiratory distress syndrome, ARDS) role. Methods The A549 cells in logarithmic growth phase were treated with different concentrations of Netrin-1 (0, 0.5, 5, 50 and 500 μg / L) for 12 h. A549 cells were divided into two groups: drug group (500μg / LNetrin-1), inhibition group (1μmol / L PSB1115 + 500μg / L Netrin-1) and control group (RPMI1640 medium only) The transcription level of α-ENaC mRNA in each group was detected by RT-PCR. The expression of α-ENaC protein in each group was detected by Western blot. Results After treated with Netrin-1 for 6 h, the mRNA and protein expressions of α-ENaC mRNA in A549 cells were significantly higher than those in the control group (P <0.05) at the same concentration for 6 h. At the same time, The relative transcription level and relative expression of α-ENaC mRNA were significantly higher than those of the control group (P <0.05) when the concentration of Netrin-1 was more than 5μg / L, and the dose-dependent manner (P <0.05). The mRNA and protein expressions of α-ENaC mRNA and protein in drug group were significantly higher than those in control group and control group (P <0.05). PSB1115 significantly inhibited the transcription and protein expression of α-ENaC mRNA. Conclusion Netrin-1 can upregulate the expression of α-ENaC gene through adenosine A2b receptor pathway, which is beneficial to the prognosis of ALI and ARDS.