本文關(guān)鍵詞： 百草枯 纖維化 血小板反應(yīng)蛋白-1 CD47 氧化應(yīng)激　出處：《川北醫(yī)學(xué)院》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:以人源性A549細(xì)胞為研究對象,從體外構(gòu)建百草枯(Paraquat,PQ)致肺纖維化模型,初步探討TSP-1及其受體CD47在PQ致肺纖維化中的作用及其可能的發(fā)病機(jī)制。方法:體外培養(yǎng)人A549細(xì)胞,首先分為正常組對照組、PQ組、空白對照組,采用CCK-8法檢測不同濃度(50μM、100μM、200μM、400μM、600μM、800μM、1000μM)的PQ刺激不同時(shí)間(12h、24h、48h)對細(xì)胞存活率的影響,以細(xì)胞半數(shù)存活率為標(biāo)準(zhǔn)篩選出作用于后續(xù)試驗(yàn)的濃度和時(shí)間點(diǎn)。此后分為正常組、PQ組,PQ組下設(shè)1個(gè)時(shí)間點(diǎn)(24h)和3個(gè)濃度(50μM、100μM、200μM)。倒置顯微鏡下觀察細(xì)胞形態(tài)變化;采用酶聯(lián)免疫吸附(Enzyme Linked Immunosorbent Assay,ELISA)法測定纖維連接蛋白(Fibronectin,FN)、I型膠原的相對表達(dá)量;免疫細(xì)胞化學(xué)(Immunocytochemistry,ICC)法觀察TSP-1、CD47的蛋白表達(dá)情況;免疫熒光雙染(Immunofluorescence double staining)觀察TSP-1、CD47共表達(dá);實(shí)時(shí)熒光定量PCR(Real Time PCR,RT-PCR)檢測TSP-1、CD47mRNA的表達(dá)量,Western印跡法(Western Blot,WB)檢測TSP-1、CD47蛋白表達(dá);流式細(xì)胞儀檢測細(xì)胞內(nèi)活性氧(Reactive Oxygen Species,ROS)水平。最后,再分為正常組、PQ組、Anti-TSP1組(PQ+TSP1中和抗體),PQ組下設(shè)1個(gè)時(shí)間點(diǎn)(24h)和1個(gè)濃度(200μM);Anti-TSP1組(24h)加入TSP1中10μg/ml與200μM的PQ共培養(yǎng)。再次觀察細(xì)胞存活率、細(xì)胞形態(tài)、TSP-1和CD47基因和蛋白水平、細(xì)胞內(nèi)ROS水平和FN、I型膠原表達(dá)變化。結(jié)果:1.PQ作用時(shí)間一定時(shí),細(xì)胞活力隨PQ濃度增加而降低,呈時(shí)間濃度依賴性;與正常組相比,除50μM的PQ作用12h時(shí)的細(xì)胞的活力下降沒有統(tǒng)計(jì)學(xué)意義(P0.05)外,其余均有統(tǒng)計(jì)學(xué)差異(P0.05);2.正常組細(xì)胞連接緊密,形態(tài)均一,呈鵝卵石狀,排列整齊,島狀或成團(tuán)生長,貼壁強(qiáng)、增殖能力強(qiáng);PQ組細(xì)胞隨著PQ濃度增加,細(xì)胞間隙逐漸增大,形態(tài)逐漸變得不均一、無規(guī)則,伸出不規(guī)則突起,呈紡錘體形或長梭形,排列紊亂,增殖能力逐漸減弱;3.隨著PQ作用濃度增加,與正常組相比,PQ組FN、I型膠原的相對表達(dá)量逐漸增多,呈濃度依賴性,以200μM時(shí)最為明顯;4.隨著PQ作用濃度增加,與正常組相比,PQ刺激組TSP-1、CD47 mRNA水平表達(dá)上調(diào)、蛋白表達(dá)量明顯增加,以200μM時(shí)最為明顯;5.與正常組相比,PQ組TSP-1、CD47蛋白表達(dá)量明顯增加,均以200μM時(shí)最為明顯;TSP-1、CD47在細(xì)胞質(zhì)中存在共表達(dá);6.與正常組相比,ROS水平在24h時(shí)明顯升高。加入TSP1中和抗體后,與PQ組相比,Anti-TSP1組細(xì)胞活力明顯增加,形態(tài)向正常細(xì)胞形態(tài)變化;TSP-1、CD47的mRNA水平及蛋白表達(dá)量下降;ROS的過表達(dá)受到抑制;FN、I型膠原的相對表達(dá)量減少。結(jié)論:1.200μM的PQ刺激A549細(xì)胞24h能夠成功構(gòu)建體外肺纖維化模型;2.PQ刺激A549細(xì)胞后可誘導(dǎo)TSP-1和CD47的表達(dá),且在細(xì)胞質(zhì)中存在共表達(dá);3.PQ可刺激A549細(xì)胞發(fā)生氧化應(yīng)激;4.TSP-1中和抗體可下調(diào)TSP-1、CD47 mRNA水平及蛋白表達(dá)量,抑制氧化應(yīng)激狀態(tài),降低FN、I型膠原表達(dá)量;5.TSP-1-CD47可能通過誘導(dǎo)氧化應(yīng)激參與了PQ致肺纖維化過程。
[Abstract]:Objective: to establish a model of pulmonary fibrosis induced by Paraquat PQ from human A549 cells in vitro. To explore the role of TSP-1 and its receptor CD47 in PQ induced pulmonary fibrosis and its possible pathogenesis. Methods: human A549 cells were cultured in vitro and divided into normal control group and PQ group. In the blank control group, the different concentrations of 50 渭 M ~ 100 渭 m ~ (100 渭 m) ~ (100 渭 m) ~ (100 渭 m) ~ (100 渭 m) were detected by CCK-8 method. The effect of PQ (1 000 渭 M) on cell survival was observed at different time points (12h ~ 24h ~ 48h). The concentration and time point acting on the follow-up test were selected according to the standard of 50% cell survival rate. After that, they were divided into normal group (PQ group) and three concentration groups (50 渭 M), one time point (24 h) and three concentrations (50 渭 M) under PQ group. The morphologic changes of the cells were observed under inverted microscope. Enzyme Linked Immunosorbent Assay was used as enzyme linked immunosorbent assay (Elisa). The relative expression of fibronectinine fibronectin (FN) type I collagen was determined by ELISAmethod. The expression of TSP-1mCD47 protein was observed by immunocytochemistry-ICCassay. Immunofluorescence double staining was used to observe the co-expression of TSP-1 and CD47. The expression of TSP-1 and CD47 mRNA was detected by real-time fluorescence quantitative PCR(Real Time PCR-RT-PCR. Western blot was used to detect the expression of TSP-1 and CD47 protein. Flow cytometry (FCM) was used to detect the level of reactive Oxygen species in the cells. Finally, it was divided into normal group and PQ group. In the Anti-TSP1 group, the concentration of PQ TSP1 neutralizing antibody was 24 h) and the concentration of PQ was 200 渭 m. Anti-TSP1 group was co-cultured with 10 渭 g / ml of TSP1 and 200 渭 M PQ for 24 h. Cell survival rate and cell morphology were observed again. The changes of TSP-1 and CD47 gene and protein levels, ROS level and type I collagen expression in the cells were observed. Results the cell viability decreased with the increase of PQ concentration at the same time. In a time-concentration dependent manner; Compared with the normal group, the activity of the cells treated with 50 渭 M PQ for 12 h was not significantly decreased (P0.05), but there was a significant difference in the other groups. 2. In the normal group, the cells were closely connected, uniform in shape, cobbled, arranged neatly, island or agglomerate, strong adherent and strong proliferative ability; In PQ group, with the increase of PQ concentration, the cell gap gradually increased, the morphology became uneven, irregular protruding, spindle-shaped or long fusiform, disordered arrangement, and the proliferation ability gradually weakened. 3. With the increase of PQ concentration, the relative expression of FNNI collagen in PQ group gradually increased in a concentration dependent manner, especially at 200 渭 M. 4. With the increase of PQ concentration, the expression of TSP-1 + CD47 mRNA was up-regulated and the protein expression was significantly increased in PQ stimulated group, especially at 200 渭 M. 5. The expression of TSP-1 and CD47 protein in PQ group was significantly higher than that in normal group, especially at 200 渭 M. There was coexpression of TSP-1 and CD47 in cytoplasm. 6. Compared with the control group, the level of Ros increased significantly at 24 h. After adding TSP1 neutralizing antibody, the cell viability of Anti-TSP1 group was significantly higher than that of PQ group. Morphologic changes to normal cells; The mRNA level and protein expression of TSP-1 CD47 were decreased. The overexpression of ROS was inhibited. Conclusion the lung fibrosis model of A549 cells can be successfully constructed by 1. 200 渭 M PQ stimulation for 24 h. 2. PQ could induce the expression of TSP-1 and CD47 in A549 cells, and there was coexpression in the cytoplasm of A549 cells. 3. PQ stimulated oxidative stress in A549 cells. 4. TSP-1 neutralizing antibody could down-regulate the expression of TSP-1 CD47 mRNA and protein, inhibit oxidative stress and decrease the expression of FNN-1 collagen. 5. TSP-1-CD47 may be involved in the process of pulmonary fibrosis induced by PQ by inducing oxidative stress.
【學(xué)位授予單位】：川北醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R595.4;R563

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