钙激活Cl^-通道蛋白在急性呼吸窘迫综合征患者体外气道上皮细胞中的表达及其作用实验研究

Expression and function of CLCA in ARDS airway epithelial cells in vitro

目的:探讨钙激活Cl^-通道蛋白(CLCA)在急性呼吸窘迫综合征(ARDS)体外气道上皮细胞中的蛋白表达情况,并初步探讨其作用。方法:运用脂多糖(LPS)刺激16HBE细胞系,Western Blot检测hCLCA1蛋白表达水平与ARDS的时程关系;运用hCLCA1-siRNA干扰16HBE细胞系中hCLCA1的表达,real-time RT-PCR法检测细胞系中hCLCA1的mRNA和蛋白表达水平,验证siRNA是否对目的基因成功抑制。同时检测炎症因子的mRNA表达变化水平,观察CLCA的降低与炎症因子表达的关系。结果:Western Blot结果显示:16HBE细胞系加LPS(1μg/ml)刺激12h、24h后hCLCA1的蛋白表达量下降,且LPS刺激24h后hCLCA1的降低有统计学差异(P<0.05)。real-time RT-PCR检测证实hCLCA1-siRNA对目的基因成功抑制,表明成功构建hCLCA1低表达的16HBE细胞系;同时real-time RT-PCR结果显示TNF-α和IL-1β的mRNA表达水平升高(P<0.05)。结论:CLCA在LPS刺激后的培养HBE细胞系中蛋白表达水平下降,并表现为时间依赖性。抑制HBE细胞系hCLCA1的基因表达,则该细胞分泌炎症因子的水平升高,推测CLCA在ARDS体外气道上皮细胞分泌炎症介质过程中发挥负性调节作用。

Objective:To explore the protein expression level of CLCA in ARDS airway epithelial cells in vitro and to explore its role.Methods:Western Blot was performed to detect the protein levels of hCLCA1 in 16-HBE cell lines stimulated LPS.Real-time RT-PCR was used to test the mRNA and the protein expression levels of hCLCA1 in 16-HBE cell lines,which were transfected hCLCA1-Si-RNA. Real-time RT-PCR was used to examine the mRNA expression levels ofinflammatory factors in 16-HBE cell lines,and the relationship between the decrease of CLCA and the expression of inflammatory factors was observed.Results:Western Blot results showed that the protein expression of hCLCA1 decreased at 12 h and 24 h after the stimulation of 16 HBE cell lines with LPS(1μg/ml), and the decrease of hCLCA1 was statistically significant at 24 h after LPS stimulation(P<0.05).Real-time RT-PCR result confirmed that there was a significant reduce in both the mRNA and the protein expression levels of hCLCA1 in 16-HBE cell lines transfected hCLCA1-Si-RNA,revealling a successful suppressed of hCLCA1 in 16-HBE cell lines.And Real-time RT-PCR results showed that the mRNA expression levels of TNF-α and IL-1β increased obviously in 16-HBE cell lines(P<0.05).Conclusion:The protein expression level of CLCA decreased in the 16 HBE cell lines with LPS, and the decreased level was more obvious with the extension of time.The suppression of hCLCA1 can increase the mRNA expression levels of inflammatory cytokines in HBE cell lines,suggesting that CLCA secreted by airway goblet cells inhibit the inflammatory response in the process of ARDS.