本文選題：百草枯 + 肺纖維化　； 參考：《西南醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:構(gòu)建體外百草枯(paraquat,PQ)細(xì)胞纖維化模型,觀察PQ對(duì)II型肺泡上皮細(xì)胞(A549細(xì)胞)中解整合素-金屬蛋白酶17(a disintegrin and metalloproteinase-17,ADAM17)表達(dá)的影響和探討ADAM17在PQ中毒致肺纖維化中的作用。方法:1.建立細(xì)胞纖維化模型:1)體外培養(yǎng)A549細(xì)胞,將對(duì)數(shù)生長(zhǎng)期的A549細(xì)胞分為正常組、PQ組(50、100、200、400、600、800、1000umol/L),分別作用12h、24h、36h、48h,應(yīng)用CCK-8法檢測(cè)細(xì)胞存活率,篩選出理想的PQ濃度(50、100、200umol/L)和作用時(shí)間(12h、24h、48h)。2)將對(duì)數(shù)生長(zhǎng)期的A549細(xì)胞分為正常組、PQ組(50、100、200umol/L),作用24h,應(yīng)用倒置相差顯微鏡觀察細(xì)胞形態(tài),采用酶聯(lián)免疫吸附法(Enzyme linked immunosorbent assay,ELISA)測(cè)定各組纖維化標(biāo)志物I型膠原(type I collagen,Col I)和纖連蛋白(fibronectin,FN)的表達(dá),以確認(rèn)纖維化模型的成功。2.觀察ADAM17在本細(xì)胞模型中的表達(dá):將A549細(xì)胞分為正常組、PQ組(PQ濃度50、100、200umol/L),作用12h、24h、48h,采用(reverse transcription-polymerase chain reaction,RT-PCR)、免疫細(xì)胞化學(xué)(immunocytochemistry,ICC)及蛋白免疫印跡(Western Blot,WB)分別檢測(cè)ADAM17 m RNA和蛋白的表達(dá)。3.ADAM17在PQ中毒致肺纖維化中的作用探討:使用ADAM17抑制劑(tumor necrosis factor-αprocessing inhibitor-1,TAPI-1),初步探討ADAM17在PQ中毒細(xì)胞模型中的作用;以TAPI-1進(jìn)行干預(yù),將對(duì)數(shù)生長(zhǎng)期的A549細(xì)胞分為正常組、PQ組(200umol/L)、PQ+TAPI-1組(PQ 200umol/L+TAPI-1 20umol/L),作用時(shí)間24h,RT-PCR、ICC及Western blot分別檢測(cè)ADAM17 m RNA及蛋白的表達(dá)情況,以CCK-8法檢測(cè)各組細(xì)胞活力,倒置相差顯微鏡觀察細(xì)胞形態(tài),ELISA測(cè)定各組Col I、FN的表達(dá)量。結(jié)果:1.隨著PQ濃度增加及作用時(shí)間延長(zhǎng),A549細(xì)胞活性呈下降趨勢(shì)(P0.05),呈濃度和時(shí)間依賴性。2.倒置顯微鏡下正常的A549細(xì)胞融合呈鋪路石樣生長(zhǎng),排列比較緊密,經(jīng)PQ誘導(dǎo)后細(xì)胞排列較松散,細(xì)胞間連接變疏松,部分細(xì)胞溶解、死亡。3.ELISA結(jié)果顯示,隨PQ濃度增加,Col I和FN表達(dá)增強(qiáng)(P0.05),成功建立PQ誘導(dǎo)的細(xì)胞纖維化模型。4.百草枯細(xì)胞纖維化模型中有ADAM17的表達(dá):1)ICC顯示,ADAM17在A549細(xì)胞胞漿表達(dá),隨PQ濃度增加,黃色陽性顆粒沉積越多。2)RT-PCR和Western blot表明,隨著PQ濃度增加,ADAM17 m RNA及蛋白水平表達(dá)明顯增加(P0.05);隨著PQ作用時(shí)間延長(zhǎng),ADAM17 m RNA及蛋白水平表達(dá)增加(P0.05),在24h達(dá)到高峰,呈一定時(shí)間依賴性。5.TAPI-1抑制ADAM17的表達(dá)和纖維化:1)ICC顯示,PQ+TAPI-1組細(xì)胞質(zhì)中黃色陽性顆粒的沉積較PQ組減少。2)RT-PCR及Western blot表明,PQ+TAPI-1組較PQ組相比,ADAM17 m RNA及蛋白表達(dá)水平均下調(diào)(P0.05)。3)CCK-8結(jié)果提示PQ+TAPI-1組細(xì)胞活力較PQ組有所改善(P0.05)。4)顯微鏡下觀察PQ+TAPI-1組細(xì)胞形態(tài)及細(xì)胞間隙介于正常組和PQ組之間,細(xì)胞溶解較PQ組減少。5)ELISA結(jié)果顯示,PQ+TAPI-1組較PQ組Col I和FN的表達(dá)下降(P0.05)。結(jié)論:1.百草枯構(gòu)建體外A549細(xì)胞纖維化模型。2.百草枯對(duì)A549細(xì)胞的毒性作用具有濃度和時(shí)間依賴性。3.ADAM17在百草枯誘導(dǎo)的A549細(xì)胞中過表達(dá),可能參與了百草枯誘導(dǎo)的肺纖維化過程。4.TAPI-1通過抑制ADAM17的表達(dá),從而達(dá)到抑制百草枯肺纖維化的作用。
[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.
【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R595.4;R563

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