大剂量百草枯致肺泡上皮A549细胞凋亡的研究

Study of apoptosis in aveolar epithial A549 cells exposed to high-dose paraquat

目的通过大剂量百草枯诱导A549细胞的凋亡，观察上皮标记物E-cadherin、间质标记物q-SMA、凋亡指标caspase-8、caspase-3、caspase-9等的表达，探讨凋亡的可能机制。方法先用不同剂量的百草枯（0．1～1．0mmol／L）溶液与A549细胞共培养24h及48h，观察细胞形态，MTT法分别测细胞存活率。后分别用0．1、0．2、0．3mmol／L百草枯溶液与A549细胞共培养24h，行RT-PCR检测α-SMk、E-cadherin、caspase-8、caspase-3及caspase-9在不同浓度下的mRN＆的表达量。然后用0．1、0．2、0．3mmol／L百草枯溶液与A549细胞共培养24h，行Western-blot检测α-SMA、E-cadherin、caspase-8、caspase-3及caspase-9的蛋白表达情况。结果A549细胞与百草枯溶液共培养24h后，随着浓度的增加，细胞的存活率明显降低，差异有统计学意义（P〈0．05）。培养48h后，细胞存活率降低更为明显（P〈0．05）。随百草枯浓度的增大和时间的延长，细胞形态学变化也更为显著。A549细胞与0．1、0．2和0．3mmol／L浓度的百草枯共培养24h。经RT-PCR及Western-blot检测，α-SMA及E-cadherin的mRNA表达量及蛋白表达量随浓度的增加而降低，差异具有统计学意义（P〈0．05），而caspase-8、caspase-9、caspase-3的mRNA及蛋白表达量随浓度的增加而升高（P〈0．05）。结论短时间大剂量的百草枯中毒后，A549细胞的存活率明显降低，细胞形态发生显著变化，E-cadherin、α-SMA的表达量减少。而caspase-8以及下游因子caspase-9和caspase-3明显升高。

Objective To We Used high-dose paraquat to induce apoptosis of A549 cells, and observed the expression of E-cadherin,α-SMA, caspase - 8, caspase-3 and caspase-9. Then explored the possible mechanisms of apoptosis. Methods A549 cells were treated with various concentrations of PQ （0. 1-1.0 mmol/L） for 24 and 48 hours, then observe the cell morphology and measured cell survival rate respectively determined by MTr method. A549 cells were treated with various concentrations of PQ （0. 1, 0. 2, 0. 3 mmol/L ） for 24 hours. The expressions of the mRNAs for α-SMA, E-cadherin, caspase-8, caspase-3 and caspase-9 were analysed by quantitative real-time PCR. Exposed to various concentrations of PQ （0. 1 , 0. 2, 0. 3mmol/L） for 24 hours, we analysed the protein expressions of α-SMA, E-cadherin, caspase-8, caspase-3 and caspase-9 by Western blot analysis. Results Treated with various concentrations of PQ （0. 1-1.0mmol/L ） for 24 hours, the A549 cells survival rate significantly reduced by MTY method. For 48 hours, the survival rate was lower. So, in subsequent experiments, we used 0. 1, 0. 2 and0. 3 mmol/L concentration of paraquat to A549 cells, trained for 24 hours. After high-dose （0. 1, 0. 2 and 0. 3 mmol/L） exposure to PQ, a decrease in E-cadherin was observed while a decrease in α-SMA was also detected of the mRNAs. While the expressions of the mRNAs for caspase-8, caspase-3 and caspase-9 were increase. The protein expressions of α-SMA and E-cadherin were decrease with the increase of concentrations by Western blot analysis, and the levels of caspase-8, caspase-3 and caspase-9 were increase. Conclusions After high-dose short-time exposure to PQ, the survival rate of A549 cells is decreased obviously, cell morphology changed significantly, and the expressions of α-SMA and E-cadherin were decrease. But the expressions of caspase-8, caspase-3 and caspase-9 were increase.