【摘要】：納米二氧化硅(silicon dioxide,SiO_2)俗稱白炭黑,因特殊的理化性.質(zhì)被廣泛應(yīng)用于食品、涂料、橡膠、化妝品和醫(yī)藥衛(wèi)生等許多領(lǐng)域,享有“工業(yè)味精”之美譽(yù)。中國是世界白炭黑生產(chǎn)大國之一,隨著納米SiO_2應(yīng)用的日益廣泛,相關(guān)行業(yè)人群接觸的機(jī)會越來越多,其潛在危害不容忽視。動物實(shí)驗(yàn)研究表明,納米SiO_2經(jīng)呼吸道進(jìn)入體內(nèi)后可引起大/小鼠肺部炎癥反應(yīng)和肺組織纖維化等。納米Si02致肺纖維化作用的機(jī)制目前尚未完全明了。上皮間充質(zhì)轉(zhuǎn)化(epithelial-to-messenchymal,EMT)是肺纖維化進(jìn)程中的重要環(huán)節(jié)。已有研究報道m(xù)iRNAs的異常表達(dá)與EMT的發(fā)生過程有關(guān),Let-7d、miR-192、miR-124、miR-26a、miR-10b、miR-487a等可能通過TGF-β1信號通路在EMT的發(fā)生發(fā)展過程中發(fā)揮重要的調(diào)控作用。TGF-β1可以通過依賴Smads的方式誘導(dǎo)EMT,刺激肺上皮細(xì)胞轉(zhuǎn)化為肺間質(zhì)細(xì)胞,促進(jìn)肺纖維化的發(fā)生。本研究在前期納米Si02經(jīng)一次性氣管灌注大鼠染塵60、90天時主要表現(xiàn)為肺間質(zhì)纖維化的基礎(chǔ)上,以高通量測序顯示miR-702-3p為納米SiO_2致大鼠肺組織纖維化中差異表達(dá)上調(diào)的miRNA為前提,經(jīng)生物功能學(xué)分析發(fā)現(xiàn),miR-702-3p 可能與轉(zhuǎn)化生長因子-β1(transforming growth factor-β1,TGF-β1)通路相關(guān)。miR-702-3p 可能通過預(yù)測靶基因骨形態(tài)發(fā)生蛋白-7(bone morphogenetic protein-7,BMP-7)調(diào)控TGF-β1信號通路發(fā)揮調(diào)節(jié)EMT的作用從而參與納米SiO_2致肺纖維化的發(fā)生發(fā)展過程。為闡明miR-702-3p在納米SiO_2致大鼠肺組織纖維化中的生物調(diào)節(jié)作用及明確miR-702-3p、TGF-β1信號通路、EMT三者之間的關(guān)系,研究從整體動物和細(xì)胞兩個水平來探討相關(guān)分子機(jī)制,實(shí)驗(yàn)結(jié)果如下:1.在線數(shù)據(jù)庫TargetScan預(yù)測miR-702-3p的靶基因,其可能靶基因共136個。GO和KEGG通路分析這些靶基因的生物功能學(xué),發(fā)現(xiàn)miR-702-3p對TGF-β信號通路和Smad蛋白信號轉(zhuǎn)導(dǎo)中有調(diào)控作用,本研究將TGF-β超家族成員之一的BMP-7作為miR-702-3p重要的預(yù)測靶基因,探索miR-702-3p參與調(diào)控納米SiO_2誘導(dǎo)EMT的相關(guān)機(jī)制。2.實(shí)時熒光定量PCR(qRT-PCR)檢測結(jié)果顯示,25mg/mL納米Si02染塵60d后大鼠肺組織中miR-702-3p 表達(dá)是對照組的 2.04 倍(P0.05);BMP-7、TGF-β1、Smad3、E 鈣粘蛋白(E-cadherin,E-cad)、α-平滑肌肌動蛋白(α-smooth muscle acti,α-SMA)、Ⅲ型膠原蛋白(Collagen type Ⅲ,COL3)mRNA 的表達(dá)分別為對照組的0.40、1.28、1.71、0.57、2.70、1.53倍,差異均有統(tǒng)計學(xué)意義(P0.05);蛋白免疫印跡法(Western Blot)檢查結(jié)果表明,納米SiO_2染塵組大鼠肺組織中BMP-7、TGF-β1、Smad3、E-cad、α-SMA、COL3蛋白表達(dá)量是對照組的0.60、1.48、1.24、0.65、1.43、1.23倍,差異均有統(tǒng)計學(xué)意義(P0.05)。雙熒光素酶報告基因檢測結(jié)果顯示,將野生型BMP-7的3'UTR的熒光素酶報告質(zhì)粒載體分別與miR-702-3p mimic/NC 共轉(zhuǎn)染于人肺腺癌上皮細(xì)胞系(A549 細(xì)胞),miR-702-3p mimic 組與 miR-702-3p NC組的報告熒光值分別為0.73±0.010、1.00±0.005,差異有統(tǒng)計學(xué)意義(P0.05);將BMP-7突變位點(diǎn)的載體質(zhì)粒再分別與miR-702-3p mimic/NC共轉(zhuǎn)染于A549細(xì)胞,miR-702-3p mimic組與miR-702-3p NC組的報告熒光值分別為0.98±0.060、1.00±0.016,差異無統(tǒng)計學(xué)意義(P0.05)。結(jié)果提示納米SiO_2致大鼠肺間質(zhì)纖維化過程中發(fā)生了 EMT,miR-702-3p與BMP-7可通過CCACCCG-GGUGGGC的位點(diǎn)結(jié)合,靶向抑制BMP-7的表達(dá)并激活TGF-β1信號通路,促進(jìn)肺上皮細(xì)胞轉(zhuǎn)化為肺間質(zhì)細(xì)胞,產(chǎn)生膠原蛋白增加,發(fā)生肺纖維化。3.納米SiO_2對大鼠肺泡巨噬細(xì)胞(NR8383細(xì)胞)染塵24h,四甲基偶氮唑藍(lán)法(MTT)檢測得其IC_(50)為 0.761 mg/mL,95%可信區(qū)間為 0.001～3.844 mg/mL。50μg/mL 納米 Si02 染毒 NR8383 細(xì)胞 24h 后細(xì)胞的存活率在85%以上,酶聯(lián)免疫吸附(ELISA)法檢測納米SiO_2染毒NR8383細(xì)胞24h上清液中TGF-β1含量為1612.67±15.32pg/mL,與陰性對照組1072.67±60.10pg/mL比較,差異有統(tǒng)計學(xué)意義(P0.05);納米SiO_2染毒組NR8383細(xì)胞中miR-702-3p的表達(dá)水平是對照組的3.11倍(P0.05),BMP-7 mRNA與蛋白表達(dá)水平分別是對照組的0.39和0.30倍(P0.05)。50μg/mL納米Si02染毒NR8383細(xì)胞24h后的上清液與A549細(xì)胞共培養(yǎng)24h后,空白對照組、染毒NR8383細(xì)胞上清液組A549細(xì)胞的增殖率分別為(100±6)%、(101±5)%,差異無統(tǒng)計學(xué)意義(P0.05);染毒組A549細(xì)胞中miR-702-3p的表達(dá)是對照組的 1.22 倍(P0.05);BMP-7、TGF-β1、Smad3、E-cad、α-SMA、COL3 mRNA 表達(dá)水平分別是對照組的 0.25、1.33、1.53、0.99、6.75、2.68 倍,差異有統(tǒng)計學(xué)意義(P0.05,E-cad 的 P0.05)。納米 Si02染毒組A549細(xì)胞中BMP-7、TGF-β1、Smad3、E-cad、α-SMA、COL3蛋白表達(dá)水平分別是對照組的0.48、1.88、2.20、0.40、1.91、3.1 倍,差異有統(tǒng)計學(xué)意義(P0.05)。miR-702-3p mimic/mimic NC、miR-702-3p inhibitor/inhibitor NC 和空白對照組分別轉(zhuǎn)染 NR8383 細(xì)胞48h 后,各組增殖率分別為(101.40±3.45)%、(100.03±2.91)%、(100.76±0.96)%、(92.37±3.26)%、(100±3.89)%,差異無統(tǒng)計學(xué)意義(P0.05),鏡下未觀察到細(xì)胞有明顯變化;轉(zhuǎn)染48h后miR-702-3p mimic轉(zhuǎn)染組的miR-702-3p的表達(dá)水平是陰性對照組的27.78倍(差異倍數(shù)≥2,P0.05),表明miR-702-3p mimic轉(zhuǎn)染成功;miR-702-3p inhibitor轉(zhuǎn)染NR8383細(xì)胞48h后miR-702-3p的表達(dá)水平是陰性對照組的0.19倍(P0.05),miR-702-3p mimic轉(zhuǎn)染組NR8383細(xì)胞中BMP-7mRNA與蛋白的表達(dá)水平分別是陰性對照組的1.07、0.43 倍(P0.05),miR-702-3p inhibitor轉(zhuǎn)染組NR8383 細(xì)胞中BMP-7mRNA與蛋白的表達(dá)水平分別是陰性對照組的2.02、1.32倍(P0.05);miR-702-3pmimic轉(zhuǎn)染組NR8383細(xì)胞中TGF-β1mRNA與蛋白的表達(dá)水平分別是陰性對照組的1.23、1.58倍(P0.05),miR-702-3p inhibitor轉(zhuǎn)染組NR8383細(xì)胞中TGF-β1 mRNA與蛋白的表達(dá)水平分別是陰性對照組的0.52、0.63倍(P0.05),提示轉(zhuǎn)染miR-702-3p可通過抑制BMP-7的表達(dá),促進(jìn)TGF-β1生成增加。TGF-β1、TGF-β1+TGF-β1受體阻斷劑分別誘導(dǎo)A549細(xì)胞48h后,對照組、TGF-β1+RB組與TGF-β1組的細(xì)胞增殖率分別為(100±5.04%)、(106.28±1.38)%、(108.99±6.51)%,差異無統(tǒng)計學(xué)意義(P0.05);5ng/mL的TGF-β1誘導(dǎo)A549細(xì)胞48h后,觀察到細(xì)胞從上皮樣逐漸轉(zhuǎn)變?yōu)槌衫w維細(xì)胞樣,qRT-PCR檢測結(jié)果顯示,TGF-β1 誘導(dǎo)組 A549 細(xì)胞中 miR-702-3p、BMP-7、Smad3、E-cad、α-SMA、COL3mRNA 的表達(dá)水平分別是對照組的1.37、0.39、0.57、0.18、3.14、2.64倍(P0.05),TGF-β1誘導(dǎo)組A549細(xì)胞中BMP-7、Smad3、E-cad、α-SMA、COL3 蛋白的表達(dá)量分別是對照組的 0.41、1.82、0.72、1.61、14.51 倍(P0.05);TGF-β1受體阻斷劑誘導(dǎo)3h后再給與TGF-β1誘導(dǎo)48h后A549細(xì)胞的形態(tài)未發(fā)生明顯改變,BMP-7蛋白表達(dá)增加(1.68倍)外miR-702-3p、Smad3、E-cad、α-SMA、COL3mRNA與蛋白表達(dá)水平均未出現(xiàn)明顯變化(P0.05)。提示TGF-β1對miR-702-3p有上調(diào)作用,促使EMT的發(fā)生。綜上所述,納米SiO_2致大鼠肺間質(zhì)纖維化過程中發(fā)生了 EMT,BMP-7是miR-702-3p的靶基因。miR-702-3p、TGF-β1信號通路與EMT過程密切相關(guān),一方面miR-702-3p與BMP-7可通過CCACCCG-GGUGGGC的位點(diǎn)結(jié)合,抑制靶基因BMP-7的表達(dá),活化TGF-β1信號通路,促使肺上皮細(xì)胞向肺間質(zhì)細(xì)胞轉(zhuǎn)化,膠原蛋白分泌增多,促進(jìn)肺纖維化;另一方面TGF-β1可上調(diào)miR-702-3p的表達(dá),促進(jìn)EMT過程。
[Abstract]:Nano-silica (SiO_2), commonly known as silica, is widely used in food, coatings, rubber, cosmetics and medical hygiene and many other fields, enjoying the reputation of "industrial monosodium glutamate". Animal studies have shown that nano-SiO_2 can cause pulmonary inflammation and fibrosis in rats and mice after it enters the body through the respiratory tract. The mechanism of pulmonary fibrosis induced by nano-SiO_2 has not been fully understood. It has been reported that the abnormal expression of microRNAs is related to the occurrence of EMT. Let-7d, microRNAs-192, microRNAs-124, microRNAs-26a, microRNAs-10b, microRNAs-487a may play an important regulatory role in the development of EMT through TGF-beta 1 signaling pathway. TGF-beta 1 can induce EMT and stimulate EMT by relying on Smads. Pulmonary epithelial cells were transformed into pulmonary interstitial cells to promote the development of pulmonary fibrosis. On the basis of the primary manifestation of pulmonary interstitial fibrosis in rats exposed to nano-Si02 by one-off tracheal perfusion for 60,90 days, the high-throughput sequencing showed that microRNAs-702-3p were differentially expressed and up-regulated in rat pulmonary fibrosis induced by nano-SiO_2. Biological function analysis revealed that microRNAs-702-3p may be involved in the transforming growth factor-beta 1 (TGF-beta 1) pathway. MicroRNAs-702-3p may play a role in regulating EMT by predicting the target gene bone morphogenetic protein-7 (BMP-7) regulating the TGF-beta 1 signaling pathway. To elucidate the role of microRNAs-702-3p in the regulation of pulmonary fibrosis induced by nano-SiO_2 in rats and to clarify the relationship between microRNAs-702-3p, TGF-beta 1 signaling pathway and EMT, we studied the molecular mechanism of microRNAs-702-3p at both animal and cellular levels. The results were as follows: 1. TargetS online database. Mi-702-3p target genes were predicted by can. A total of 136 probable target genes were analyzed by GO and KEGG pathways. It was found that Mi-702-3p could regulate the TGF-beta signaling pathway and signal transduction of Smad protein. BMP-7, one of the members of TGF-beta superfamily, was used as an important predictive target gene of Mi-702-3p in this study. Real-time fluorescence quantitative PCR (qRT-PCR) results showed that the expression of microRNAs-702-3p in lung tissues of rats exposed to 25 mg/mL nano-Si02 for 60 days was 2.04 times higher than that of control group (P 0.05); BMP-7, TGF-beta 1, Smad3, E-cadherin, alpha-smooth muscle actin (alpha-smooth muscle, alpha-smooth muscle, alpha-smooth muscle actini, alpha-smooth muscle actin) were detected. The expression of SMA and COL3 mRNA was 0.40, 1.28, 1.71, 0.57, 2.70 and 1.53 times higher than that of the control group (P 0.05). The expression of BMP-7, TGF-beta 1, Smad3, E-cad, alpha-SMA and COL3 protein in lung tissue of rats exposed to nano-SiO_2 was significantly higher than that of the control group (P 0.05). The results of double luciferase reporter gene test showed that the luciferase reporter plasmid vector of wild type BMP-7 was co-transfected with Mi-702-3p mimic/NC into human lung adenocarcinoma epithelial cell line (A549 cells), Mi-702-3p mic group and Mi-702-3, respectively. The reported fluorescence values of P NC group were 0.73 (+ 0.010) and 1.00 (+ 0.005) respectively, and the difference was statistically significant (P 0.05); the vector plasmid of BMP-7 mutation site was transfected into A549 cells with Mi-702-3p mimic/NC, respectively. The reported fluorescence values of Mi-702-3p mic group and Mi-702-3p NC group were 0.98 (+ 0.060) and 1.00 (+ 0.016), respectively, with no significant difference (P 0.05). The results suggest that EMT occurs in the process of pulmonary interstitial fibrosis induced by nano-SiO_2 in rats. Mi-702-3p and BMP-7 can bind to the site of CCACCCG-GGUGGC, inhibit the expression of BMP-7 and activate the TGF-beta 1 signaling pathway, promote the transformation of pulmonary epithelial cells into pulmonary interstitial cells, increase the production of collagen and induce pulmonary fibrosis in rats. Alveolar macrophages (NR8383 cells) were exposed to dust for 24 hours, and their IC_ (50) was 0.761 mg/mL by MTT assay. The 95% confidence interval was 0.001-3.844 mg/mL. The survival rate of NR8383 cells exposed to nano-Si02 was above 85% after 24 hours. The supernatant of NR8383 cells exposed to nano-SiO_2 was detected by enzyme linked immunosorbent assay (ELISA) for 24 hours. The content of TGF-beta 1 in the solution was 1612.67+15.32 pg/mL, which was significantly different from that in the negative control group (P 0.05). The expression of microRNA-702-3p in NR8383 cells of nano-SiO_2 group was 3.11 times higher than that of the control group (P 0.05), and the expression of BMP-7 mRNA and protein were 0.39 and 0.30 times higher than that of the control group (P 0.05). The proliferation rate of A549 cells in NR8383 cell supernatant group was (100 65 The expression levels of BMP-7, TGF-beta 1, Smad3, E-cad, alpha-SMA, COL3 in A549 cells were 0.48, 1.88, 2.20, 0.40, 1.91, 3.1 times higher than those in control group (P 0.05, P 0.05 for E-cad). 5. After 48 hours of transfection, the proliferation rates of NR8383 cells were (101.40 (3.45)%, (100.03 (2.91)%, (100.76 (0.96)%, (92.37 (3.26)%, (100 (3.89)%, respectively. There was no significant difference (P 0.05). After 48 hours of transfection, no significant changes were observed in NR8383 cells. The expression level of microRNAs-702-3p in the microRNAs-702-3p mimic transfection group was 27.78 times higher than that in the negative control group (P 0.05), indicating that the microRNAs-702-3p mimic transfection was successful; the expression level of microRNAs-702-3p inhibitor in NR8383 cells 48 hours after microRNAs-702-3p inhibitor transfection was 0.19 times higher than that in the negative control group (P 0.05), BMP-7 mRNA and BMP-7 mRNA in NR8383 cells of the microRNAs-702-3p mic transfection group were 0.19 times higher than those in the negative control group (P 0. The expression levels of BMP-7 mRNA and protein in NR8383 cells transfected with miR-702-3p inhibitor were 2.02 and 1.32 times higher than those in the negative control group (P 0.05), and the expression levels of TGF-beta 1 mRNA and protein in NR8383 cells transfected with miR-702-3pmic were 1.23 times higher than those in the negative control group (P 0.05). TGF-1 mRNA and protein expression levels in NR8383 cells transfected with microRNAs-702-3p inhibitor were 0.52 and 0.63 times higher than those in the negative control group (P 0.05), suggesting that microRNAs-702-3p could inhibit the expression of BMP-7 and promote the production of TGF-beta 1. TGF-1, TGF-beta 1 + TGF-beta 1 receptor blockers induced A549 cells 48 hours later in the control group, respectively. The cell proliferation rates of GF-beta 1+RB group and TGF-beta 1 group were (100+5.04%), (106.28+1.38)%, (108.99+6.51)%. There was no significant difference between them (P 0.05). After 48h induction of A549 cells by 5 ng/ml TGF-beta 1, the cells gradually transformed from epithelial to fibroblast-like cells. The results of qRT-PCR showed that the cells of TGF-beta 1 induced A549 cells were microRNA702-3p, 3 P. The expression levels of BMP-7, Smad3, E-cad, alpha-SMA, COL3 mRNA were 1.37, 0.39, 0.57, 0.18, 3.14, 2.64 times as high as those in the control group (P 0.05). The expression levels of BMP-7, Smad3, E-cad, alpha-SMA, COL3 protein in A549 cells induced by TGF-beta 1 receptor blocker were 0.41, 1.82, 0.72, 1.61, 14.51 times as high as those in the control group (P 0.05). The expression of BMP-7 protein increased (1.68 times) and the mRNA and protein levels of extracellular microarray-702-3p, Smad3, E-cad, alpha-SMA, COL3 did not change significantly (P 0.05). These results suggest that TGF-beta 1 can up-regulate the expression of microarray-702-3p and promote the occurrence of EMT in rat pulmonary fibrosis induced by nano-SiO_2. Mi-702-3p, TGF-beta 1 signaling pathway is closely related to EMT process. On the one hand, Mi-702-3p and BMP-7 can bind to the site of CCACCCG-GGUGGC, inhibit the expression of target gene BMP-7, activate TGF-beta 1 signaling pathway, promote the transformation of lung epithelial cells into pulmonary interstitial cells, increase the secretion of collagen, and promote the proliferation of lung epithelial cells. Pulmonary fibrosis; on the other hand, TGF- beta 1 can increase the expression of miR-702-3p and promote the EMT process.
【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R135.2

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8 耿開興;肺纖維化60例臨床分析[J];中原醫(yī)刊;2003年12期

9 何平平;張平;;局部腎素-血管緊張素-醛固酮系統(tǒng)與肺纖維化[J];醫(yī)學(xué)綜述;2007年09期

10 張永勝;馮一中;顧振綸;;藥物防治肺纖維化的研究進(jìn)展[J];中國血液流變學(xué)雜志;2007年01期

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8 楊s，

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