摘要
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.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2006年第10期883-889,共7页
Chinese Journal of Microbiology and Immunology
基金
广西高校百名中青年学科带头人资助计划[桂教人(2005)64号]
广西高校病理学科人才小高地创新团队计划[桂教人(2005)81号]
关键词
气道平滑肌细胞
磷脂酰肌醇3激酶
核因子-κB
增殖
凋亡
转化生长因子Β1
Airway smooth muscle cells( ASMC)
Phosphoinositide 3-kinases(PI3-K)
Nuclear factor kappa B(NF-κB)
Proliferation
Apoptosis
Transforming growth factor-beta 1(TGF-β1 )