尘螨经PI3K通路对人支气管上皮细胞和血管内皮生长因子表达的影响及可能的调控机制

Effect of dust mites on the expression of human bronchial epithelial cells and vascular endothelial growth factor through PI3K pathway and its possible regulatory mechanism

目的探讨尘螨经PI3K通路对人支气管上皮细胞(16-HBE)和血管内皮生长因子(VEGF)表达的影响及可能的调控机制。方法培养16-HBE细胞,根据不同尘螨刺激浓度和10μmol/LLY294002分为正常对照组、尘螨100 U/L组、尘螨200 U/L组、尘螨400 U/L组、尘螨600 U/L组、尘螨800 U/L组、尘螨1000 U/L组、尘螨2000 U/L组、LY294002+尘螨400 U/L组、LY294002组。采用四唑盐(MTT)比色法检测不同尘螨浓度的16-HBE活力,再检测LY294002组、LY294002+尘螨400 U/L组及正常对照组16-HBE活力,采用荧光定量聚合酶链式反应(RT-PCR)测量不同条件刺激下16-HBE的VEGF mRNA表达水平,采用酶联免疫吸附实验法(ELISA)检测不同条件刺激下16-HBE的VEGF蛋白水平。结果尘螨2000 U/L组细胞活力低于正常对照组、尘螨100 U/L组、尘螨200 U/L组、尘螨400 U/L组、尘螨600U/L组、尘螨800U/L组及尘螨1000 U/L组(P<0.05)。LY294002组、LY294002+尘螨400 U/L组、正常对照组16-HBE活力比较差异无统计学意义。正常对照组VEGF(165)mRNA表达水平低于尘螨200 U/L组、尘螨400 U/L组、尘螨600 U/L组、尘螨800 U/L组、尘螨1000 U/L组、LY294002+尘螨400 U/L组,尘螨200U/L组低于尘螨400U/L组,高于LY294002+尘螨400U/L组、LY294002组,尘螨400U/L组高于尘螨600U/L组、尘螨800U/L组、尘螨1000 U/L组、LY294002+尘螨400 U/L组、LY294002组,尘螨600 U/L组、尘螨800 U/L组、尘螨1000 U/L组均高于LY294002+尘螨400 U/L组、LY294002组,尘螨400 U/L+LY294002组高于LY294002组(P<0.05)。正常对照组16-HBE培养上清VEGF蛋白水平低于尘螨200 U/L组、尘螨400 U/L组、尘螨600 U/L组、尘螨800U/L组、尘螨1000U/L组、尘螨400U/L+LY294002组,尘螨200 U/L组低于尘螨600 U/L组,高于尘螨1000 U/L组、LY294002+尘螨400 U/L组、LY294002组,尘螨400 U/L组高于尘螨600 U/L组、尘螨800 U/L组、尘螨1000 U/L组、LY294002+尘螨400 U/L组、LY294002组,尘螨600 U/L组、尘螨800 U/L组均高于尘螨1000 U/L组、LY294002+尘螨400 U/L组、LY294002组,尘螨1000 U/L组、LY294002+尘螨400 U/L组均高于LY294002组(P<0.05)。结论尘螨经PI3K通路16-HBE在VEGF mRNA和蛋白水平上的高表达,其机制可能是经由PI3K与哮喘密切相关信号通路,为尘螨导致哮喘的可能机制提供新线索,可为哮喘的治疗带来新思路。

Objective To investigate the effect of dust mites on the expression of human bronchial epithelial cells(16-HBE)and vascular endo-thelial growth factor(VEGF)through PI3K pathway and its possible regulatory mechanism.Methods 16-HBE cells were cultured and divided into normal control group,dust mite 100 U/L group,dust mite 200 U/L group,dust mite 400 U/L group,dust mite 600 U/L group,dust mite 800 U/L group,dust mite 1000 U/L group,dust mite 2000 U/L group,LY294002+dust mite 400 U/L group and LY294002 group according to different con-centrations of dust mite stimulation and 10μmol/L LY294002.Methyltetrazolium(MTT)assay was used to detect the activity of 16-HBE at different concentrations of dust mite,and then the activity of 16-HBE in the LY294002 group,the LY294002+dust mite 400 U/L group and normal control group was detected,the expression level of VEGF mRNA in 16-HBE under different conditions was measured by fluorescence quantitative poly-merase chain reaction(RT-PCR),and the expression level of VEGF protein in 16-HBE under different conditions was detected by enzyme-linked im-munosorbent assay(ELISA).Results The cell viability in the dust mite 2000 U/L group was lower than that in the normal control group,the dust mite 100 U/L group,dust mite 200 U/L group,dust mite 400 U/L group,dust mite 600 U/L group,dust mite 800 U/L group and dust mite 1000 U/L group(P<0.05).There was no significant difference in 16-HBE activity among the LY294002 group,LY294002+dust mite 400 U/L group and nor-mal control group.The expression level of VEGF(165)mRNA in the normal control group was lower than that in the dust mite 200 U/L group,dust mite 400 U/L group,dust mite 600 U/L group,dust mite 800 U/L group,dust mite 1000 U/L group,LY294002+dust mite 400 U/L group,dust mite 200 U/L group was lower than the dust mite 400 U/L group,higher than LY294002+dust mite 400 U/L group and LY294002 group.The dust mite 400 U/L group was higher than the dust mite 600 U/L group,the dust mite 800 U/L group,the dust mite 1000 U/L group,the LY294002+dust mite 400 U/L group,the LY294002 group,the dust mite 600 U/L group,the dust mite 800 U/L group,the dust mite 1000 U/L group were higher than the LY294002+dust mite 400 U/L group,the LY294002 group,the dust mite 400 U/L+LY294002 group was higher than the LY294002 group(P<0.05).The VEGF protein level in 16-HBE culture supernatant in the normal control group was lower than that of dust mite 200 U/L group,dust mite 400 U/L group,dust mite 600 U/L group,dust mite 800 U/L group,dust mite 1000 U/L group,dust mite 400 U/L+LY294002 group,dust mite 200 U/L group was lower than dust mite 600 U/L group,higher than dust mite 1000 U/L group,LY294002+dust mite 400 U/L group,LY294002 group,dust mite 400 U/L group was higher than dust mite 600 U/L group,dust mite 800 U/L group,dust mite 1000 U/L group,LY294002+dust mite 400 U/L group and LY294002 group,dust mite 600 U/L group and dust mite 800 U/L group were higher than dust mite 1000 U/L group,LY294002+dust mite 400 U/L group and LY294002 group,dust mite 1000 U/L group and LY294002+dust mite 400 U/L group were higher than the LY294002 group(P<0.05).Conclusion The high expression of 16-HBE in VEGF gene and protein levels through PI3K pathway may be closely related to asthma through PI3K signaling pathway,which provides new clues for the possible mechanism of asthma caused by dust mites and brings new ideas for the treatment of asthma.