PM2.5暴露通过调节miR-21表达诱发血管内皮通透性改变
发布时间：2018-08-18 16:53
【摘要】：目的:血管内皮通透性改变不会引起显著的病变,但通透性的增大可能导致诸如冠心病、动脉粥样硬化等血管疾病,甚至癌症。文献报道血管内皮通透性改变是血管疾病发生的初始环节,通透性的增大导致血管弹性变化,血液中脂质也会沉积到血管内皮下,最终导致血管变硬、增厚,诱发一系列的血管病变。本研究旨在研究PM2.5暴露后其在体内外诱发血管内皮通透性改变,通过研究血管内皮通透性增大的机制,阐明机制与炎症因子、转录因子、microRNA相关,为阐述PM2.5诱发血管内皮通透性增大的机制研究提供强有力的证据,为未来应对PM2.5诱发血管内皮通透性改变进而引起血管疾病提供治疗的新思路。方法:利用CCK-8检测不同浓度不同暴露时间下HUVECs活性;通过Transwell检测单层HUVECs通透性;利用基因芯片、Quantitative Real-time PCR检测差异表达microRNA;通过PCR扩增、酶切酶连技术构建质粒;通过生物信息学分析、双荧光素酶报告基因实验、ChIP验证TIMP3 3’UTR存在miR-21的靶位点、p-STAT3靶向结合miR-21启动子;采用气管滴注的方法构建SD大鼠实验模型,通过检测伊文思蓝渗漏情况证明SD大鼠血管内皮通透性改变;利用Western Blot、HE染色、免疫组化、ELISA技术检测PM2.5暴露下炎症因子、转录因子、血管内皮通透性相关蛋白的变化;通过mi R-21抑制剂干扰,探究PM2.5诱发血管内皮通透性增大的信号通路。结果:PM2.5暴露后,HUVECs的活性降低,具有剂量和浓度依赖性;单层HUVECs通透性增大,并具有时间依赖性;基因芯片结果显示PM2.5暴露能引起多个microRNA转录差异,其中miR-21转录差异最明显;生物信息学预测和双报告实验证实了miR-21靶向结合TIMP3 3’UTR;Western blot和miR-21抑制剂的转染发现TIMP3的表达受miR-21的调控,而作为受TIMP3抑制的MMP9活性随之改变,直接影响血管内皮通透性;PM2.5暴露能引起炎症因子IL-6的增多,而IL-6的增多又导致了p-STAT3的上调;生物信息学预测、双报告实验和染色质免疫沉淀实验证实了p-STAT3能靶向结合miR-21启动子,并引起其转录上调;PM2.5暴露后动物模型体内炎症因子、转录因子、miR-21、通透性相关蛋白的检测也同样显示出PM2.5暴露引起血管内皮通透性增大,且与IL-6/p-STAT3/mir-21/TIMP3/MMP9相关。结论:通过体内外实验发现PM2.5暴露引起氧化应激反应,激活IL-6/STAT3通路,促使p-STAT3靶向结合miR-21启动子并促进其转录,而miR-21的上调通过抑制其靶序列TIMP3的表达,解除了对MMP9的拮抗作用,最终水解了血管内皮细胞外基质导致血管内皮通透性增大。
[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.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54
本文编号：2190088
[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.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54
【参考文献】
相关期刊论文 前2条
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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