ADAM17在百草枯致肺纤维化中的初步研究

发布时间：2018-06-20 19:05

本文选题：百草枯 + 肺纤维化　；参考：《西南医科大学》2017年硕士论文

【摘要】：目的:构建体外百草枯(paraquat,PQ)细胞纤维化模型,观察PQ对II型肺泡上皮细胞(A549细胞)中解整合素-金属蛋白酶17(a disintegrin and metalloproteinase-17,ADAM17)表达的影响和探讨ADAM17在PQ中毒致肺纤维化中的作用。方法:1.建立细胞纤维化模型:1)体外培养A549细胞,将对数生长期的A549细胞分为正常组、PQ组(50、100、200、400、600、800、1000umol/L),分别作用12h、24h、36h、48h,应用CCK-8法检测细胞存活率,筛选出理想的PQ浓度(50、100、200umol/L)和作用时间(12h、24h、48h)。2)将对数生长期的A549细胞分为正常组、PQ组(50、100、200umol/L),作用24h,应用倒置相差显微镜观察细胞形态,采用酶联免疫吸附法(Enzyme linked immunosorbent assay,ELISA)测定各组纤维化标志物I型胶原(type I collagen,Col I)和纤连蛋白(fibronectin,FN)的表达,以确认纤维化模型的成功。2.观察ADAM17在本细胞模型中的表达:将A549细胞分为正常组、PQ组(PQ浓度50、100、200umol/L),作用12h、24h、48h,采用(reverse transcription-polymerase chain reaction,RT-PCR)、免疫细胞化学(immunocytochemistry,ICC)及蛋白免疫印迹(Western Blot,WB)分别检测ADAM17 m RNA和蛋白的表达。3.ADAM17在PQ中毒致肺纤维化中的作用探讨:使用ADAM17抑制剂(tumor necrosis factor-αprocessing inhibitor-1,TAPI-1),初步探讨ADAM17在PQ中毒细胞模型中的作用;以TAPI-1进行干预,将对数生长期的A549细胞分为正常组、PQ组(200umol/L)、PQ+TAPI-1组(PQ 200umol/L+TAPI-1 20umol/L),作用时间24h,RT-PCR、ICC及Western blot分别检测ADAM17 m RNA及蛋白的表达情况,以CCK-8法检测各组细胞活力,倒置相差显微镜观察细胞形态,ELISA测定各组Col I、FN的表达量。结果:1.随着PQ浓度增加及作用时间延长,A549细胞活性呈下降趋势(P0.05),呈浓度和时间依赖性。2.倒置显微镜下正常的A549细胞融合呈铺路石样生长,排列比较紧密,经PQ诱导后细胞排列较松散,细胞间连接变疏松,部分细胞溶解、死亡。3.ELISA结果显示,随PQ浓度增加,Col I和FN表达增强(P0.05),成功建立PQ诱导的细胞纤维化模型。4.百草枯细胞纤维化模型中有ADAM17的表达:1)ICC显示,ADAM17在A549细胞胞浆表达,随PQ浓度增加,黄色阳性颗粒沉积越多。2)RT-PCR和Western blot表明,随着PQ浓度增加,ADAM17 m RNA及蛋白水平表达明显增加(P0.05);随着PQ作用时间延长,ADAM17 m RNA及蛋白水平表达增加(P0.05),在24h达到高峰,呈一定时间依赖性。5.TAPI-1抑制ADAM17的表达和纤维化:1)ICC显示,PQ+TAPI-1组细胞质中黄色阳性颗粒的沉积较PQ组减少。2)RT-PCR及Western blot表明,PQ+TAPI-1组较PQ组相比,ADAM17 m RNA及蛋白表达水平均下调(P0.05)。3)CCK-8结果提示PQ+TAPI-1组细胞活力较PQ组有所改善(P0.05)。4)显微镜下观察PQ+TAPI-1组细胞形态及细胞间隙介于正常组和PQ组之间,细胞溶解较PQ组减少。5)ELISA结果显示,PQ+TAPI-1组较PQ组Col I和FN的表达下降(P0.05)。结论:1.百草枯构建体外A549细胞纤维化模型。2.百草枯对A549细胞的毒性作用具有浓度和时间依赖性。3.ADAM17在百草枯诱导的A549细胞中过表达,可能参与了百草枯诱导的肺纤维化过程。4.TAPI-1通过抑制ADAM17的表达,从而达到抑制百草枯肺纤维化的作用。
[Abstract]:Objective: to construct an in vitro paraquat (PQ) cell fibrosis model, and to observe the effect of PQ on the expression of integrin metal protease 17 (a disintegrin and metalloproteinase-17, ADAM17) in II type alveolar epithelial cells (A549 cells) and to explore the role of ADAM17 in the pulmonary fibrosis induced by PQ poisoning. Method: 1. the model of cell fibrosis was established: 1) A549 cells were cultured in vitro, and the logarithmic growth period A549 cells were divided into normal group, PQ group (501002004006008001000umol/L), 12h, 24h, 36h, 48h, and CCK-8 method was used to detect the cell survival rate, and the ideal PQ concentration (50100200umol/L) and action time were selected to divide the logarithmic growth period into the normal group. Group (50100200umol/L), acting 24h, using inverted phase contrast microscope to observe cell morphology, and using Enzyme linked immunosorbent assay (ELISA) to determine the expression of I type collagen (type I collagen, Col) and fibronectin in each group of fibrosis markers to confirm the success of the fibrosis model The expression in this cell model: A549 cells were divided into normal group, PQ group (PQ concentration 50100200umol/L), 12h, 24h, 48h, reverse transcription-polymerase chain reaction, RT-PCR, immunocytochemistry and protein expression respectively. The role of ADAM17 in pulmonary fibrosis induced by PQ poisoning: using the ADAM17 inhibitor (tumor necrosis factor- alpha processing inhibitor-1, TAPI-1), the role of ADAM17 in the PQ poisoned cell model was preliminarily discussed. 1 20umol/L), the expression of ADAM17 m RNA and protein were detected by 24h, RT-PCR, ICC and Western blot respectively. The cell viability was detected by CCK-8 method, and the cell morphology was observed by inverted phase contrast microscope. P0.05), under the concentration and time dependent.2. inversion microscope, normal A549 cells fused with paving stone like growth. The cells were arranged more closely. After PQ induction, the cells were loosely arranged, the intercellular connections became loose, and some cells dissolved. The death.3.ELISA results showed that the Col I and FN expression enhanced with the increase of PQ concentration (P0.05), and PQ induction was successfully established. The expression of ADAM17 in the fibrotic model of.4. paraquat cells in the cell fibrosis model: 1) ICC showed that ADAM17 was expressed in the cytoplasm of A549 cells, the more.2 was deposited with the concentration of PQ, the more.2 RT-PCR and Western blot showed that the expression of ADAM17 and egg white increased with the increase of PQ concentration. The expression of ADAM17 m RNA and protein increased (P0.05), reached the peak at 24h, and a certain time dependent.5.TAPI-1 inhibited the expression and fibrosis of ADAM17. 1) ICC showed that the deposition of yellow positive particles in the cytoplasm of the PQ+TAPI-1 group was less.2 than that of the PQ group. The average down-regulation (P0.05).3) CCK-8 results showed that the cell viability of PQ+TAPI-1 group was better than that of PQ group (P0.05).4) under microscope, the cell morphology and intercellular space of PQ+TAPI-1 group were between the normal group and the PQ group, and the cell dissolution was lower than that of the PQ group. The ELISA results showed that the expression of the group was lower than that of the PQ group. Conclusion: 1. paraquat was compared. In vitro A549 cell fibrosis model.2. paraquat has a toxic effect on A549 cells with concentration and time dependent.3.ADAM17 over expression in A549 cells induced by paraquat, which may be involved in the process of pulmonary fibrosis induced by paraquat,.4.TAPI-1 can inhibit the fibrosis of paraquat by inhibiting the expression of ADAM17.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R595.4;R563

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