CGA衍生多肽CHR抑制肿瘤坏死因子-α诱导血管内皮细胞高渗透性的钙相关信号机制探讨

发布时间：2018-01-24 07:54

本文关键词： 内皮细胞 EA.hy926细胞钙嗜铬粒蛋白A Chromofungin 肿瘤坏死因子-α　出处：《重庆医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的：探讨嗜铬粒蛋白A (Chromogranin A, CGA)衍生多肽Chromofungin (CHR)抑制肿瘤坏死因子-α (TNF-α)诱导血管内皮细胞高渗透性的钙相关信号机制。方法：以人脐静脉内皮细胞系EA.hy926细胞为研究对象,CHR、 TNF-α为处理因素。1)利用过氧辣根氧化物(HRP)穿透Transwell小室吸光度(A)值表示EA.hy926细胞渗透性变化；2)利用细胞免疫荧光法、激光共聚焦显微镜观察肌动蛋白F-actin表达和分布；3)利用激光共聚焦显微镜检测EA.hy926田胞内钙离子浓度([Ca2+]i)的变化。结果：1)CHR抑制TNF-a诱导的血管内皮细胞渗透性增加。与对照组相比,TNF-a组明显增加EA.hy926田胞渗透性(1.20±0.09 vs 1.77±0.20, P0.05); CHR (10nmol/L、100nmol/L和1000 nmol/L)抑制TNF-a诱导的EA.hy926细胞渗透性增加,其中CHR (10 nmol/L和100 nmol/L)显著降低TNF-a诱导的EA.hy926细胞高渗透性(1.77±0.20 vs 1.32±0.13, P0.05; 1.77±0.20 vs 1.24±0.18, P0.05), CHR (1000 nmol/L)能够降低TNF-a诱导的EA.hy926细胞高渗透性,但差异无统计学意义(1.77±0.20 vs 1.41±0.26, P0.1)。2) CHR抑制TNF-a诱导的血管内皮细胞渗透性相关蛋白的表达变化和分布异常。与对照组相比,TNF-a引起F-actin排列紊乱,应力纤维形成增多；CHR明显改善TNF-a诱导的上述效应。3)CHR抑制TNF-a诱导的血管内皮细胞外钙内流。CHR (10 nmol/L、100 nmol/L和1000 nmol/L)作用于EA.hy926细胞时细胞内Ca2+浓度无明显变化。TNF-a诱导EA.hy926细胞外Ca2+快速内流,各时间点细胞内Ca2+荧光强度Ft40、 Ft100与起始荧光值Ft30相比,具有显著性差异(荧光强度分别为：41.50±0.55 vs 56.19±0.84, P0.01; 41.500±0.55 vs 53.46±0.93, P0.01), CHR (10 nmol/L、100 nmol/L和1000 nmol/L)能够抑制TNF-α引起的Ca2+快速内流,CHR (100 nmol/L)抑制效果最显著。结论：CHR抑制TNF-α诱导的EA.hy926细胞渗透性增加,这一作用可能是通过阻止TNF-α诱导的细胞外Ca2+内流来实现。
[Abstract]:Objective: to study Chromogranin A (Chromogranin A). CGA-derived peptide Chromofungin) inhibits tumor necrosis factor- 伪 (TNF- 伪). Methods: human umbilical vein endothelial cell line EA.hy926 cells were studied. CHR, TNF- 伪 as the processing factor. 1) HRP (peroxyhorseradish oxide) was used to penetrate the Transwell chamber absorbance (A) value to indicate the change of EA.hy926 cell permeability. 2) the expression and distribution of actin F-actin were observed by laser confocal microscopy and immunofluorescence assay. 3) the intracellular Ca ~ (2 +) concentration in EA.hy926 field was detected by laser confocal microscopy (. [Results: 1: 1 chr inhibited the increase of vascular endothelial cell permeability induced by TNF-a, compared with the control group. In TNF-a group, the cell permeability in EA.hy926 field was significantly increased by 1.20 卤0.09 vs 1.77 卤0.20, P 0.05; CHR 10nmol / L, 100nmol / L and 1000nmol / L inhibited the increase of EA.hy926 cell permeability induced by TNF-a. Among them, 10 nmol/L and 100 nmol / L CHR significantly decreased the high permeability of EA.hy926 cells induced by TNF-a (. 1.77 卤0.20 vs 1.32 卤0.13. P0.05; 1.77 卤0.20 vs 1.24 卤0.18, P 0.05). CHR (1000nmol / L) could reduce the high permeability of EA.hy926 cells induced by TNF-a. But the difference was not statistically significant (1.77 卤0.20 vs 1.41 卤0.26). CHR inhibited the expression and distribution of osmotic proteins in vascular endothelial cells induced by TNF-a, compared with the control group. TNF-a caused disorder of F-actin arrangement and increased formation of stress fibers. CHR significantly improved the above effects of TNF-a. 3CHR inhibited the extracellular calcium influx of vascular endothelial cells induced by TNF-a for 10 nmol/L. 100 nmol/L and 1000nmol / L). There was no significant change in intracellular Ca2 concentration in EA.hy926 cells. TNF-a induced rapid Ca2 influx out of EA.hy926 cells. The intracellular Ca2 fluorescence intensity Ft40 and Ft100 were compared with the initial fluorescence value (Ft30) at each time point. There was significant difference (fluorescence intensity was: 41.50 卤0.55 vs 56.19 卤0.84, P 0.01); 41.500 卤0.55 vs 53.46 卤0.93, P 0.01, CHR 10 nmol/L. 100 nmol/L and 1000nmol / L) could inhibit the rapid influx of Ca2 induced by TNF- 伪. Conclusion CHR can inhibit the increase of TNF- 伪 -induced permeability of EA.hy926 cells. This effect may be achieved by blocking the extracellular Ca2 influx induced by TNF- 伪.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R459.7

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