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PM2.5暴露通過(guò)調(diào)節(jié)miR-21表達(dá)誘發(fā)血管內(nèi)皮通透性改變

發(fā)布時(shí)間:2018-08-18 16:53
【摘要】:目的:血管內(nèi)皮通透性改變不會(huì)引起顯著的病變,但通透性的增大可能導(dǎo)致諸如冠心病、動(dòng)脈粥樣硬化等血管疾病,甚至癌癥。文獻(xiàn)報(bào)道血管內(nèi)皮通透性改變是血管疾病發(fā)生的初始環(huán)節(jié),通透性的增大導(dǎo)致血管彈性變化,血液中脂質(zhì)也會(huì)沉積到血管內(nèi)皮下,最終導(dǎo)致血管變硬、增厚,誘發(fā)一系列的血管病變。本研究旨在研究PM2.5暴露后其在體內(nèi)外誘發(fā)血管內(nèi)皮通透性改變,通過(guò)研究血管內(nèi)皮通透性增大的機(jī)制,闡明機(jī)制與炎癥因子、轉(zhuǎn)錄因子、microRNA相關(guān),為闡述PM2.5誘發(fā)血管內(nèi)皮通透性增大的機(jī)制研究提供強(qiáng)有力的證據(jù),為未來(lái)應(yīng)對(duì)PM2.5誘發(fā)血管內(nèi)皮通透性改變進(jìn)而引起血管疾病提供治療的新思路。方法:利用CCK-8檢測(cè)不同濃度不同暴露時(shí)間下HUVECs活性;通過(guò)Transwell檢測(cè)單層HUVECs通透性;利用基因芯片、Quantitative Real-time PCR檢測(cè)差異表達(dá)microRNA;通過(guò)PCR擴(kuò)增、酶切酶連技術(shù)構(gòu)建質(zhì)粒;通過(guò)生物信息學(xué)分析、雙熒光素酶報(bào)告基因?qū)嶒?yàn)、ChIP驗(yàn)證TIMP3 3’UTR存在miR-21的靶位點(diǎn)、p-STAT3靶向結(jié)合miR-21啟動(dòng)子;采用氣管滴注的方法構(gòu)建SD大鼠實(shí)驗(yàn)?zāi)P?通過(guò)檢測(cè)伊文思藍(lán)滲漏情況證明SD大鼠血管內(nèi)皮通透性改變;利用Western Blot、HE染色、免疫組化、ELISA技術(shù)檢測(cè)PM2.5暴露下炎癥因子、轉(zhuǎn)錄因子、血管內(nèi)皮通透性相關(guān)蛋白的變化;通過(guò)mi R-21抑制劑干擾,探究PM2.5誘發(fā)血管內(nèi)皮通透性增大的信號(hào)通路。結(jié)果:PM2.5暴露后,HUVECs的活性降低,具有劑量和濃度依賴性;單層HUVECs通透性增大,并具有時(shí)間依賴性;基因芯片結(jié)果顯示PM2.5暴露能引起多個(gè)microRNA轉(zhuǎn)錄差異,其中miR-21轉(zhuǎn)錄差異最明顯;生物信息學(xué)預(yù)測(cè)和雙報(bào)告實(shí)驗(yàn)證實(shí)了miR-21靶向結(jié)合TIMP3 3’UTR;Western blot和miR-21抑制劑的轉(zhuǎn)染發(fā)現(xiàn)TIMP3的表達(dá)受miR-21的調(diào)控,而作為受TIMP3抑制的MMP9活性隨之改變,直接影響血管內(nèi)皮通透性;PM2.5暴露能引起炎癥因子IL-6的增多,而IL-6的增多又導(dǎo)致了p-STAT3的上調(diào);生物信息學(xué)預(yù)測(cè)、雙報(bào)告實(shí)驗(yàn)和染色質(zhì)免疫沉淀實(shí)驗(yàn)證實(shí)了p-STAT3能靶向結(jié)合miR-21啟動(dòng)子,并引起其轉(zhuǎn)錄上調(diào);PM2.5暴露后動(dòng)物模型體內(nèi)炎癥因子、轉(zhuǎn)錄因子、miR-21、通透性相關(guān)蛋白的檢測(cè)也同樣顯示出PM2.5暴露引起血管內(nèi)皮通透性增大,且與IL-6/p-STAT3/mir-21/TIMP3/MMP9相關(guān)。結(jié)論:通過(guò)體內(nèi)外實(shí)驗(yàn)發(fā)現(xiàn)PM2.5暴露引起氧化應(yīng)激反應(yīng),激活I(lǐng)L-6/STAT3通路,促使p-STAT3靶向結(jié)合miR-21啟動(dòng)子并促進(jìn)其轉(zhuǎn)錄,而miR-21的上調(diào)通過(guò)抑制其靶序列TIMP3的表達(dá),解除了對(duì)MMP9的拮抗作用,最終水解了血管內(nèi)皮細(xì)胞外基質(zhì)導(dǎo)致血管內(nèi)皮通透性增大。
[Abstract]:Objective: changes in vascular endothelial permeability do not cause significant lesions, but increased permeability may lead to vascular diseases such as coronary heart disease, atherosclerosis, and even cancer. It is reported that the change of vascular endothelial permeability is the initial stage of vascular disease. The increase of permeability leads to the change of vascular elasticity, and the lipid deposition in the blood will result in the hardening and thickening of blood vessels. Induce a series of vascular lesions. The purpose of this study was to study the changes of vascular endothelial permeability induced by PM2.5 in vivo and in vitro, and to elucidate the relationship between the mechanism and inflammatory factors, transcription factors and microRNAs by studying the mechanism of increased vascular endothelial permeability. In order to elucidate the mechanism of vascular endothelial permeability increase induced by PM2.5, and provide a new idea for the treatment of vascular diseases caused by PM2.5 in the future. Methods: CCK-8 was used to detect HUVECs activity at different concentrations and exposure time, Transwell was used to detect the permeability of monolayer HUVECs, gene chip quantitative Real-time PCR was used to detect differentially expressed microRNAs, PCR amplification was used to construct plasmids. By bioinformatics analysis, the double luciferase reporter gene experiment (chip) verified the existence of miR-21 target site, p-STAT3 targeted miR-21 promoter, and established an experimental model of Sprague-Dawley (SD) rats by tracheal instillation. The changes of vascular endothelial permeability in SD rats were proved by detecting the leakage of Evans blue, and the changes of inflammatory factors, transcription factors and vascular endothelial permeability related proteins under PM2.5 exposure were detected by Western blothe staining and immunohistochemistry Elisa. The signal pathway of increased vascular endothelial permeability induced by PM2.5 was explored by interference of mi R 21 inhibitor. Results the activity of HUVECs was decreased in a dose-and concentration-dependent manner, the permeability of monolayer HUVECs was increased in a time-dependent manner, and the results of gene microarray showed that PM2.5 exposure could induce multiple microRNA transcriptional differences, among which miR-21 transcriptional differences were the most significant. Bioinformatics prediction and double report experiments confirmed that the expression of TIMP3 was regulated by miR-21, and the MMP9 activity was changed as a result of TIMP3 inhibition, which was confirmed by the transfection of miR-21 targeting TIMP3 3 UTRU Western blot and miR-21 inhibitor, and the expression of TIMP3 was regulated by miR-21. Exposure to PM2.5, which directly affects vascular endothelial permeability, can lead to an increase in the inflammatory factor IL-6, while the increase of IL-6 leads to the up-regulation of p-STAT3. Bioinformatics prediction, double-report experiments and chromatin immunoprecipitation tests confirm that p-STAT3 can target miR-21 promoter. After exposure to PM2.5, the expression of inflammatory factor, transcription factor miR-21 and permeability related protein also showed that PM2.5 exposure induced increased vascular endothelial permeability, and was related to IL-6/p-STAT3/mir-21/TIMP3/MMP9. Conclusion: in vitro and in vivo, PM2.5 exposure induces oxidative stress response, activates IL-6/STAT3 pathway, promotes p-STAT3 targeting to miR-21 promoter and promotes its transcription. The up-regulation of miR-21 removes the antagonistic effect on MMP9 by inhibiting the expression of TIMP3, the target sequence of miR-21. The final hydrolysis of vascular endothelial extracellular matrix resulted in increased vascular endothelial permeability.
【學(xué)位授予單位】:廣州醫(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)】:R54

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 Stefania Oliveto;Marilena Mancino;Nicola Manfrini;Stefano Biffo;;Role of microRNAs in translation regulation and cancer[J];World Journal of Biological Chemistry;2017年01期

2 Huilian Hou;Guanjun Zhang;Hongyan Wang;Huilin Gong;Chunbao Wang;Xuebin Zhang;;High matrix metalloproteinase-9 expression induces angiogenesis and basement membrane degradation in stroke-prone spontaneously hypertensive rats after cerebral infarction[J];Neural Regeneration Research;2014年11期



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