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目的探讨体外培养的人肺腺癌细胞(A549)在百草枯(PQ)诱导下能否发生上皮-间质转化(epithelial-mesenchymal transition,EMT)及四氢吡咯二硫代氨基甲酸酯(PDTC)的干预作用和可能机制。方法体外培养A549细胞,实验分为对照A549细胞组(Control)、PQ诱导A549细胞组(PQ)、PDTC 3个浓度干预组(PQ+PDTC1,2,3;终浓度分别为10,20,40μmol/L)。用噻唑蓝(MTT)测定细胞毒性;活性氧荧光定量法(DCFH-DA)测定细胞内活性氧(ROS)水平;酶联免疫吸附试验(ELISA)测定转化生长因子β1(TGF-β1)表达水平;实时定量RCR(Real Time,RT-PCR)测定EMT相关标志物上皮细胞钙粘蛋白(Ecad)、紧密连接蛋白(ZO-1)、N-钙粘蛋白(N-cad)和α-平滑肌肌动蛋白(α-SMA)mRNA表达。结果与对照组比较,PQ(600μmol/L)作用A549细胞24h后,细胞存活率降低,细胞内ROS水平明显升高,TGF-β1表达水平明显升高,上皮细胞标志物E-cad、ZO-1基因表达下调,间质细胞标志物N-cad、α-SMA基因表达明显上调(P<0.05);PDTC干预组能够降低PQ诱导的A549细胞毒性,明显抑制上述作用,其中对TGF-β1表达、E-cad、N-cad基因表达的干预作用呈剂量依赖方式(P<0.05)。结论PQ诱导A549细胞过度表达TGF-β1,继而导致上皮细胞向间质细胞转化,这可能是PQ致肺纤维化的重要机制之一;PDTC的有效干预可能基于其抗氧化和核因子-kappaB(nuclear factor-kappaB,NF-κB)抑制作用。
Objective To investigate whether epithelial-mesenchymal transition (EMT) and pyrrolidine dithiocarbamate (PDTC) can be induced by paraquat (PQ) in cultured human lung adenocarcinoma cells (A549) ) Intervention and possible mechanisms. Methods A549 cells were cultured in vitro. The experiment was divided into control A549 cell group, PQ-induced A549 cell group (PQ) and PDTC 3 concentration intervention group (PQ + PDTC1,2,3; final concentrations were 10, 20 and 40μmol / L). Cytotoxicity was determined by MTT assay. Reactive oxygen species (ROS) was measured by reactive oxygen species-specific fluorescent quantification (DCFH-DA). Expression of transforming growth factor-β1 (TGF-β1) was measured by enzyme linked immunosorbent assay (ELISA) ; Real-time quantitative RT-PCR (RCR) was used to detect the expression of EMT related markers such as Ecad, ZO-1, N-cadherin and α- Actin (α-SMA) mRNA expression. Results Compared with the control group, A549 cells treated with PQ (600μmol / L) for 24 h decreased the cell viability and the intracellular ROS levels, while the expression of TGF-β1 was significantly increased. The expressions of epithelial cell markers E-cad and ZO- (P <0.05). PDTC intervention group could reduce the PQ-induced cytotoxicity of A549 cells and significantly inhibit the above-mentioned effect, in which the expression of TGF-β1 , E-cad, N-cad gene expression in a dose-dependent manner (P <0.05). Conclusion PQ can induce A549 cells to over-express TGF-β1, which leads to the transformation of epithelial cells into stromal cells. This may be one of the important mechanisms of PQ-induced pulmonary fibrosis. Effective intervention of PDTC may be based on its anti-oxidative and nuclear factor-kappaB nuclear factor-kappaB, NF-κB) inhibition.