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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 effects of phosphatidylinositol 3-kinase (PB-K) on the proliferation, apoptosis and the expression of transforming growth factor-β1 (TGF-β1) induced by TNF-α in rat airway smooth muscle cells. Methods Tumor necrosis factor-α (TNF-α) and PI3-K specific inhibitor wortmannin were used as a tool to inoculate TNF-α and TNF-α + wortmannin into ASMC respectively and cultured in 37 ℃ 5% CO 2 incubator . The expression of PB-K p85α and TGF-β1 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). The protein expression and localization of PI3-K p85α, PCNA and Bcl-2 were detected by immunocytochemical staining. κB activity and proliferation of ASMC were determined by MTT assay. Apoptosis was detected by Annexin V / PI double-labeled flow cytometry. The protein content of TGF-β1 was determined by ELISA. Results (1) Positive localization of cultured ASMC PB-K p85α localized in the cytoplasm. TNF-α group ASMC PI3-K p85αmRNA and protein expression levels were significantly higher than the control group and TNF-α + wortmannin group (P <0.01); (2) TNF-α group ASMC NF- (P <0.01). The NF-κB activity in TNF-α + wortmannin group was significantly lower than that in TNF-α group (P <0.01). (3) (P <0.01). There was no significant difference in apoptosis rate between TNF-αgroup and control group (P> 0.05), TNF-α The apoptosis rate of + wortmannin group was significantly higher than that of TNF-α group and control group (P <0.01). (4) The expression of PCNA and Bcl-2 protein in ASMC of TNF-αgroup was significantly higher than that in control group and TNF-α + wortmannin group (P <0.01). (5) The expression of TGF- The protein level of TGF-β1 in culture supernatant was significantly higher than that in control group and TNF-α + wortmannin group (P <0.01). Conclusion P13-K may be involved in the regulation of TNF-α-induced proliferation, apoptosis and TGF-β1 expression and secretion in rat airway smooth muscle cells. NF-κB is involved in this process as a downstream signal of PI3-K.