LncRNA-MIAT在TNF-α介导的血管炎症中的表达及作用

发布时间：2018-05-03 09:10

本文选题：长链非编码RNA + 血管炎症　；参考：《青岛大学》2017年硕士论文

【摘要】：目的:体外观察血管内皮细胞(endothelial cells,ECs)和血管平滑肌细胞(vascular smooth muscle cell,VSMC)在肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)刺激下,细胞内长链非编码RNA心肌梗死相关转录本(long non-coding RNA myocardial infarction associated transcript,LncRNA-MIAT)的表达变化,并探讨LncRNA-MIAT对ECs和VSMC炎症的调控作用。方法:以TNF-α诱导ECs和VSMC炎症状态,采用Western Blot、PCR及q RT-PCR方法分别检测ECs和VSMC炎症状态下细胞间黏附分子-1(intercellular adhesion molecule-1,ICAM-1)、血管细胞粘附分子-1(vascular cell adhesion molecule-1,VCAM-1)和LncRNA-MIAT的表达情况。应用LncRNA-MIAT的小干扰核糖核酸(si RNA MAIT)转染ECs和VSMC,观察LncRNA-MIAT敲低对ICAM-1和VCAM-1表达的影响。通过划痕实验,观察ECs和VSMC的迁移功能。结果:1.(1)内皮细胞PCR结果:与空白对照相比,加入lncRNA-MIAT引物的条带明显,提示ECs中表达LncRNA-MIAT,不同引物浓度的条带亮度的比较差异不明显,提示当引物浓度增加时,LncRNA-MIAT表达量增加不明显。(2)平滑肌细胞PCR结果:与空白对照相比,加入lncRNA-MIAT引物的条带明显,提示VSMC中表达LncRNA-MIAT,不同引物浓度的条带亮度的比较差异不明显,提示当引物浓度增加时,LncRNA-MIAT表达量增加不明显。2.(1)内皮细胞q RT-PCR结果:随着TNF-α刺激时间延长和刺激浓度增高,LncRNA-MIAT表达呈逐渐上升趋势。TNF-α刺激24 h、48 h其他刺激时长比较,lncRNA-MIAT表达量增多;TNF-α刺激浓度1.000 ng/ml、10.000 ng/ml与其他刺激浓度比较,lncRNA-MIAT表达量均增多,差异均具有统计学意义。(2)平滑肌细胞q RT-PCR结果:随着TNF-α刺激时间延长和刺激浓度增高,LncRNA-MIAT表达呈逐渐上升趋势。刺激12 h、24 h、48h其他刺激时长比较,lncRNA-MIAT表达量增多;刺激浓度10.00 ng/ml、20.000 ng/ml与其他刺激浓度比较,lncRNA-MIAT表达量均增多,差异均具有统计学意义。3.(1)内皮细胞RNA干扰结果:si RNA MAIT 48h与si RNA Scramble比较,lncRNA-MIAT表达量明显下降,差异具有统计学意义;si RNA MAIT 48 h与si RNA MAIT 24 h相比,lncRNA-MIAT表达量下降,差异具有统计学意义。(2)平滑肌细胞RNA干扰结果:si RNA MAIT 24h与si RNA Scramble比较,lncRNA-MIAT表达量明显下降,差异具有统计学意义;si RNA MAIT 48 h与si RNA Scramble比较,lncRNA-MIAT表达量明显下降,差异具有统计学意义。4.(1)内皮细胞中ICAM-1的Western Blot结果:浓度为1.000 ng/ml的TNF-α刺激ECs后,ICAM-1的表达与空白对照相比显著增高,差异有统计学意义。应用si RNA MAIT转染ECs 48 h后,应用浓度为1.000 ng/ml TNF-α刺激ECs后,与阳性对照相比,ICAM-1表达量被明显抑制,差异有统计学意义;与空白对照相比,差异无统计学意义。(2)内皮细胞中VCAM-1的Western Blot结果:浓度为1.000 ng/ml的TNF-α刺激ECs后,VCAM-1的表达与空白对照相比显著增高,差异有统计学意义。应用si RNA MAIT转染ECs 48 h后,应用浓度为1.000 ng/ml TNF-α刺激ECs后,与阳性对照相比,VCAM-1表达量被明显抑制,差异有统计学意义;与空白对照相比,差异无统计学意义。(3)平滑肌细胞中ICAM-1的Western Blot结果:浓度为10.00 ng/ml的TNF-α刺激VSMC后,ICAM-1的表达与空白对照相比显著增高,差异有统计学意义。应用si RNA MAIT转染VSMC 48 h后,应用浓度为10.00 ng/ml TNF-α刺激VSMC后,与阳性对照相比,ICAM-1表达被抑制,差异有统计学意义;与空白对照相比,差异无统计学意义。5.(1)内皮细胞划痕实验结果:浓度为1.000 ng/ml TNF-α刺激ECs培养24h后,与空白对照相比,ECs迁移距离延长,差异有统计学意义。si RNA MAIT敲低后的ECs加入浓度为1.000ng/ml TNF-α刺激ECs培养24h后,与空白对照相比,ECs迁移明显被抑制,差异有统计学意义。(2)平滑肌细胞划痕实验结果:浓度为10.00 ng/ml TNF-α刺激VSMC培养24h后,与空白对照相比,细胞迁移距离明显延长,差异有统计学意义。si RNA MAIT敲低后的VSMC加入浓度为10.00 ng/ml TNF-α刺激培养24h后,与空白对照相比,VSMC迁移明显被抑制,差异有统计学意义。结论:LncRNA-MIAT可能参与血管的炎症反应,并可能起到促炎作用。
[Abstract]:Objective: To observe in vitro endothelial cells (ECs) and vascular smooth muscle cells (vascular smooth muscle cell, VSMC) under the stimulation of tumor necrosis factor alpha (tumor necrosis factor- alpha, TNF- alpha). The expression changes of IPT, LncRNA-MIAT, and the regulatory role of LncRNA-MIAT on the inflammation of ECs and VSMC. Methods: TNF- alpha induced ECs and VSMC inflammatory states. 1 (vascular cell adhesion molecule-1, VCAM-1) and LncRNA-MIAT expression. Use LncRNA-MIAT's small interfering ribonucleic acid (Si RNA MAIT) transfection ECs and VSMC. The bands with lncRNA-MIAT primers were obvious, suggesting that LncRNA-MIAT was expressed in ECs. The difference of band brightness of different primers was not obvious. It suggested that when the concentration of primers increased, the expression of LncRNA-MIAT was not obvious. (2) PCR results of smooth muscle cells: compared with empty white, the bands with lncRNA-MIAT primers were obvious, suggesting that the bands of lncRNA-MIAT primers were obvious, suggesting that the bands of lncRNA-MIAT primers were obvious. The expression of LncRNA-MIAT in VSMC was not obvious in the band brightness of different primers. It was suggested that when the primer concentration increased, the increase of LncRNA-MIAT expression was not obvious in.2. (1) endothelial cells Q RT-PCR results: with the prolongation of TNF- alpha stimulation time and the increase of stimulation concentration, the expression of LncRNA-MIAT showed a gradual increase of.TNF- alpha stimulation 24 h, and 48 h. Compared with the time of stimulation, the expression of lncRNA-MIAT increased, the concentration of TNF- alpha was 1 ng/ml, and the expression of lncRNA-MIAT was increased with the concentration of 10 ng/ml. (2) the Q RT-PCR results of smooth muscle cells: the expression of LncRNA-MIAT increased with the prolongation of the time of TNF- alpha stimulation and the increase of the concentration of stimulation. Trend. Compared with 12 h, 24 h, and 48h other stimuli, the expression of lncRNA-MIAT increased, the concentration of stimulation was 10 ng/ml, 20 ng/ml was compared with other stimuli, lncRNA-MIAT expression increased, and the difference was statistically significant.3. (1) of RNA interference in endothelial cells: Si RNA MAIT. The difference has statistical significance, Si RNA MAIT 48 h and Si RNA MAIT 24 h, lncRNA-MIAT expression decreased, and the difference has statistical significance. (2) RNA interference results of smooth muscle cells: Si RNA Compared with amble, the expression of lncRNA-MIAT decreased significantly, and the difference was statistically significant.4. (1) the Western Blot result of ICAM-1 in endothelial cells: the expression of TNF- a with a concentration of 1 ng/ml was significantly higher than that of the blank control, and the difference was statistically significant. The application concentration was 1. After TNF- alpha was stimulated by ECs, the expression of ICAM-1 was significantly inhibited and the difference was statistically significant. The difference was not statistically significant compared with the blank control. (2) the Western Blot result of VCAM-1 in endothelial cells: the expression of VCAM-1 was significantly higher than that of the blank control, and the difference was statistically significant compared with that of the blank control. Significance. After the application of Si RNA MAIT to ECs 48 h, the concentration was 1 ng/ml TNF- A and ECs was stimulated, and the amount of VCAM-1 expression was significantly inhibited and the difference was statistically significant. The difference was not statistically significant compared with the blank control. (3) Western Blot results in the ICAM-1 of the smooth muscle cells: the concentration of 10 After MC, the expression of ICAM-1 was significantly higher than that of the blank control. The difference was statistically significant after the transfection of VSMC 48 h with Si RNA MAIT, with a concentration of 10 ng/ml TNF- a VSMC, the ICAM-1 expression was inhibited and the difference was statistically significant; there was no significant difference between the endothelial cells of.5. (1) compared with the blank control. The result of the mark experiment: after the concentration of 1 ng/ml TNF- alpha stimulated ECs culture 24h, compared with the blank control, the migration distance of ECs was prolonged, the difference was statistically significant after.Si RNA MAIT was knocked down, the concentration of ECs added to 1.000ng/ml TNF- alpha stimulated ECs culture, compared with the blank control, the migration movement was obviously inhibited, and the difference was statistically significant. (2) smooth muscle The result of cell scratch test: after the concentration of 10 ng/ml TNF- alpha stimulated VSMC culture 24h, the cell migration distance was obviously prolonged compared with the blank control, the difference was statistically significant after.Si RNA MAIT knockout concentration was 10 ng/ml TNF- alpha stimulation 24h, and the VSMC migration was obviously suppressed compared with the blank. The difference was statistically significant. Conclusion: LncRNA-MIAT may play an important role in the inflammatory reaction of blood vessels and may play a proinflammatory role.

【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54

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