磷脂酰肌醇3激酶对TNF-α诱导的大鼠气道平滑肌细胞增殖、凋亡和转化生长因子β1表达的影响

Effect of phosphoinositide 3-kinase on TNF-α induced proliferation,apoptosis and the expression of TGF-β1 in airway smooth muscle cells

目的探讨磷脂酰肌醇3激酶(PB-K)对TNF-α诱导的大鼠气道平滑肌细胞(ASMC)增殖、凋亡和转化生长因子β1(TGF-β1)表达的影响。方法以肿瘤坏死因子-α(TNF-α)及PI3-K特异性抑制剂wortmannin作为工具药,分别将TNF-α、TNF-α+wortmannin接种ASMC,置37℃,5％CO_2培养箱中培养。逆转录聚合酶链式反应(RT-PCR)检测PB-K p85α、TGF-β1 mRNA表达,免疫细胞化学染色法检测PI3-K p85α、PCNA、Bcl-2蛋白表达及定位,Sandwich ELISA检测NF-κB活性,四甲基偶氮唑蓝(MTY)微量比色法测定ASMC增殖,Annexin V/PI双标记流式细胞仪分析法检测细胞凋亡,ELISA测定TGF-β1蛋白质含量。结果(1)培养的ASMC PB-K p85α的阳性定位在胞浆。TNF-α组ASMC PI3-K p85αmRNA及蛋白表达水平均显著高于对照组及TNF-α+wortmannin组(P＜0．01)；(2)TNF-α组ASMC NF-κB活性与对照组相比显著增高(P＜0．01),TNF-α+wortmannin组NF-κB活性与TNF-α组相比显著降低(P＜0．01)；(3)TNF-α组ASMC的增殖反应与对照组及TNF-α+wortmannin组相比显著增加(P＜0．01)；TNF-α组细胞凋亡率与对照组相比差异无统计学意义(P＞0．05),TNF-α+wortmannin组细胞凋亡率显著高于TNF-α组及对照组(P＜0．01)。(4)TNF-α组ASMC的PCNA、Bcl-2蛋白表达量均显著高于对照组及TNF-α+wortmannin组(P＜0．01)；(5)TNF-α组ASMC的TGF-β1的mRNA表达水平、培养液上清TGF-β1的蛋白质含量均显著高于对照组及TNF-α+wortmannin组(P＜0．01)。结论P13-K可能参与调控TNF-α诱导的大鼠气道平滑肌细胞的增殖、凋亡及TGF-β1的表达和分泌,NF-κB作为PI3-K的下游信号参与此过程。

Objective To investigate the effect of phosphoinositide 3-kinase（PI3-K） on proliferation, apoptosis and the expression of TGF-β1 in airway smooth muscle cells（ASMCs）. Methods Cultured ASMCs were divided into three groups and stimulated with or without TNF-α and wortmannin in vitro. Reverse transcriptasepolymerase chain reaction（RT-PCR） was used to detect the expression of PD-K p85α and TGF-β1 mRNA, the location and protein expression of PI3-K p85α, PCNA and Bcl-2 were examined by immtmocytocbemical staining, the activity of NF-κB was detected by ELISA, the proliferation of ASMC was observed by MTr minim colorimetry techniques, the apoptosis of ASMC was analyzed by Annexin V/PI double marked flow cytometry. Results （ 1 ） The expression of PI3-K p85α was mainly located in cytoplasm. The expression of PI3-K p85α protein and mRNA of ASMC in the TNF-α group were significantly higher than that in the control group. （2） The activity of NF-κB in the TNF-α group was significantly higher than that in the control group. （3） The proliferation of ASMC in the TNF-α group was significantly higher than the control group and the TNF-α plus wortmannin group, respectively （ P 〈 0.01）. There was no significant difference in the apoptosis rate of ASMC between the TNF-α group and the controlgroup respectively（ P 〉 0.05）. The apoptosis rate of ASMC in the TNF-α plus wortmannin group was significantly higher than the TNF-α group and the control group, respectively（ P 〈 0.01 ）. （P〈0.01） .（4）The protein expression of PCNA and Bcl-2 in the TNF-α group were significantly higher than the control group and the TNF-α plus wortmannin group, respectively（ P 〈 0.01 ）. （5） The expression of TGF-β1 mRNA and the concentration of TGF-β1 protein in the TNF-α group were significantly higher than that in control group and the TNF-α plus wortmannin group, respectively（ P 〈 0. 01 Conclusion PI3-K may regulate TNF-α induced proliferation, apoptosis and the expression of TGF-β1 in ASMC, NF-κB may be involved in this process as a downstream signal of PI3-K.