IL-8介导血管平滑肌细胞迁移机制及G31P的作用初探
发布时间：2018-10-31 07:14
【摘要】：目的:动脉粥样硬化(Atherosclerosis, AS)导致的心脑血管疾病是人类健康的最大杀手,在我国的发病率逐年升高。近年来的研究表明AS是一种炎症性疾病,大量的炎症细胞和炎性因子伴随在其发生和发展过程的始终。炎症细胞参与AS表现为在损伤部位有大量炎症细胞的粘附、浸润,并且释放大量的炎性因子,趋化白细胞穿过血管内膜,诱导平滑肌细胞表型改变,继而迁移进入内膜并进行增殖。这些细胞进入内膜后,开始吞噬脂质形成泡沫细胞,加速AS的发展。趋化因子是一类具有趋化作用的小分子量分泌型蛋白,可以诱导白细胞进入血管内膜,在AS中发挥重要作用。白细胞介素-8(Interleukin-8, IL-8)是一种单核/巨噬细胞分泌的多肽,通过与其受体CXCR1/CXCR2结合从而启动中性粒细胞趋化反应。IL-8还能够诱导人主动脉血管平滑肌细胞迁移和增殖;并且可以促进AS中新生血管形成,但其具体机制尚不清楚。抑制IL-8与其受体结合为治疗AS提供了新的思路。G31P是通过对人IL-8基因的定点突变,筛选的出高亲和力,无活性的人IL-8类似物。通过实验证实了G31P可以高效的亲和CXCR1/CXCR2,对与中性粒细胞相关的感染治疗上有明显的疗效。G31P对中性粒细胞CXCR1/CXCR2的有效抑制作用能否发生在血管平滑肌细胞中尚未有研究。 方法:(1)利用RT-PCR的方法检测A7r5细胞中是否有IL-8的受体之一CXCR2的表达;(2)利用Boyden Chamber法观察IL-8和G31P对A7r5细胞迁移的影响;(3)利用划痕法和Boyden Chamber法观察G31P在IL-8诱导A7r5细胞迁移过程中的影响;(4)利用细胞免疫荧光方法观察细胞骨架变化情况;(5)利用钙离子荧光探针Fluo-3 AM检测IL-8与G31P对A7r5细胞内钙离子浓度的影响。 结果:(1)RT-PCR结果显示A7r5细胞中有CXCR2的表达;(2)IL-8诱导A7r5细胞迁移实验结果显示,随着IL-8浓度的增加,细胞迁移数呈现上升趋势,IL-8浓度为20ng/ml、50ng/ml两组的细胞迁移数较其它各组有统计学意义,但这两组之间差异无统计学意义。随着G31P浓度的增加,迁移的细胞数并未发生明显变化,各组间的差异无统计学意义。(4)划痕和迁移实验结果显示了细胞平均迁移距离与相对迁移数量两项统计值的变化情况,只用G31P处理的阴性对照组与空白对照组比较,两项统计值的变化均无统计学意义。只用IL-8诱导的阳性对照组与空白对照组比较,两项统计值的变化均有统计学意义(p0.01)。G31P抑制组与空白对照和G31P阴性对照组比较,两项统计值的变化无统计学意义;与IL-8阳性对照比较,两项统计值的变化有统计学意义(p0.01)。(5)免疫荧光实验结果显示,随着IL-8浓度增加,细胞伪足伸出明显增加,呈剂量依赖趋势。与未用G31P预处理、IL-8终浓度为20ng/ml组的细胞相比,在用G31P预处理后再用IL-8刺激,可观察到细胞伪足的伸出不明显。(6)细胞内钙离子浓度测定结果显示,随着IL-8浓度的增加(0-100ng/ml)细胞内钙离子的荧光强度逐渐增加,呈现剂量依赖趋势,与对照组相比有统计学意义。与用同样浓度的IL-8刺激组相比,用G31P预处理的细胞、再用IL-8刺激后,其细胞内钙离子的荧光强度变弱,有统计学意义。 结论:(1)IL-8可以诱导A7r5细胞的迁移、细胞伪足伸出、细胞内钙离子浓度升高,具有剂量依赖性。(2)G31P可以有效抑制IL-8对A7r5细胞的诱导作用。(3)IL-8对A7r5细胞的诱导作用可能通过调节细胞膜钙离子通道进而调节细胞功能。
[Abstract]:Objective: The cardiovascular and cerebrovascular diseases caused by atherosclerosis (AS) are the biggest killers of human health, and the incidence of cardiovascular and cerebrovascular diseases is increasing year by year in China. Recent studies have shown that AS is an inflammatory disease, and a large number of inflammatory cells and inflammatory factors are associated with the occurrence and development of AS. Inflammatory cells participate in AS expression as adhesion and infiltration of a large amount of inflammatory cells in the injured site, and release a large amount of inflammatory factors, chemotaxis the leukocytes through the intima, induce changes in the phenotype of smooth muscle cells, and then migrate into the intima and proliferate. After these cells enter the endometrium, the lipid-forming cells are phagocytized, and the development of AS is accelerated. Chemokine is a kind of small molecular weight secretory protein with chemotaxis, which can induce leukocyte to enter the intima and play an important role in AS. Interleukin-8 (Interleukin-8, IL-8) is a monocyte/ macrophage-secreted polypeptide, which initiates a neutrophil chemotaxis reaction by binding to its receptor TR1/ CD2 2. IL-8 is also capable of inducing migration and proliferation of vascular smooth muscle cells of human aorta; and it can promote neovascularization in AS, but its specific mechanism is unclear. Inhibition of IL-8 and its receptor binding provides a new way to treat AS. G3P is a high affinity, inactive human IL-8 analog by site-directed mutagenesis of human IL-8 gene. The results show that G31P can be highly effective in affinity 1/ 2 2, and has obvious therapeutic effect on the treatment of infection with neutrophils. The effective inhibitory effect of G31P on neutrophil CD1/ CD2 was not yet studied in vascular smooth muscle cells. Methods: (1) To detect the expression of IL-8 in A7r5 cells by RT-PCR; (2) observe the effect of IL-8 and G3P on the migration of A7r5 cells by Boyden Chamber method; (3) observe the effect of G3P on IL-8-induced migration of A7r5 cells by means of scratch method and Boyden Chamber method. Effects of IL-8 and G3P on intracellular calcium ion concentration in A7r5 cells were detected by means of fluorescence probe Fluo-3AM. Results: (1) The results of RT-PCR showed that the expression of cyclin 2 was found in A7r5 cells, and (2) IL-8-induced migration of A7r5 cells showed that with the increase of IL-8 concentration, the number of cell migration increased and the concentration of IL-8 was 20ng/ ml, and the number of cell migration in 50ng/ ml group was higher than that in other groups. Statistical significance, but no difference between the two groups Statistical significance. With the increase of G31P concentration, the number of migrated cells did not change significantly, and there was no difference among the groups. Statistical significance. (4) The results of scratch and migration show that the mean migration distance and the relative migration amount are two statistical values, and only the negative control group treated with G31P is compared with the blank control group. Statistical significance. Compared with blank control group, only the positive control group induced by IL-8 had statistical significance (P0.01). G31P inhibition group was compared with blank control group and G31P negative control group, and the change of two statistical values was not statistically significant. The change in the two statistical values was statistically significant compared to the sexual control (p 0. 01). (5) The results of immunofluorescence assay showed that with the increase of IL-8 concentration, the extension of pseudopodia of the cells increased significantly, and the results showed that, as the concentration of IL-8 increased, In comparison with the cells which were not pretreated with G31P, the final concentration of IL-8 was 20ng/ ml, and after pretreatment with G31P, IL-8 was used to stimulate the cells. The results showed that the fluorescence intensity of calcium ions increased gradually with the increase of IL-8 concentration (0-100ng/ ml). Statistical significance. Compared with the IL-8 stimulation group with the same concentration, the fluorescence intensity of calcium ions in the cells weakened by the stimulation of IL-8, and the fluorescence intensity of intracellular calcium ions weakened. Conclusion: (1) IL-8 can induce the migration of A7r5 cells, the extension of cells and the increase of intracellular calcium ion concentration. It is dose-dependent. (2) G31P can effectively inhibit IL-8 to A7r. Induction of 5 cells. (3) The induction of IL-8 on A7r5 cells may be mediated by the regulation of calcium ion channels in cell membranes
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
[Abstract]:Objective: The cardiovascular and cerebrovascular diseases caused by atherosclerosis (AS) are the biggest killers of human health, and the incidence of cardiovascular and cerebrovascular diseases is increasing year by year in China. Recent studies have shown that AS is an inflammatory disease, and a large number of inflammatory cells and inflammatory factors are associated with the occurrence and development of AS. Inflammatory cells participate in AS expression as adhesion and infiltration of a large amount of inflammatory cells in the injured site, and release a large amount of inflammatory factors, chemotaxis the leukocytes through the intima, induce changes in the phenotype of smooth muscle cells, and then migrate into the intima and proliferate. After these cells enter the endometrium, the lipid-forming cells are phagocytized, and the development of AS is accelerated. Chemokine is a kind of small molecular weight secretory protein with chemotaxis, which can induce leukocyte to enter the intima and play an important role in AS. Interleukin-8 (Interleukin-8, IL-8) is a monocyte/ macrophage-secreted polypeptide, which initiates a neutrophil chemotaxis reaction by binding to its receptor TR1/ CD2 2. IL-8 is also capable of inducing migration and proliferation of vascular smooth muscle cells of human aorta; and it can promote neovascularization in AS, but its specific mechanism is unclear. Inhibition of IL-8 and its receptor binding provides a new way to treat AS. G3P is a high affinity, inactive human IL-8 analog by site-directed mutagenesis of human IL-8 gene. The results show that G31P can be highly effective in affinity 1/ 2 2, and has obvious therapeutic effect on the treatment of infection with neutrophils. The effective inhibitory effect of G31P on neutrophil CD1/ CD2 was not yet studied in vascular smooth muscle cells. Methods: (1) To detect the expression of IL-8 in A7r5 cells by RT-PCR; (2) observe the effect of IL-8 and G3P on the migration of A7r5 cells by Boyden Chamber method; (3) observe the effect of G3P on IL-8-induced migration of A7r5 cells by means of scratch method and Boyden Chamber method. Effects of IL-8 and G3P on intracellular calcium ion concentration in A7r5 cells were detected by means of fluorescence probe Fluo-3AM. Results: (1) The results of RT-PCR showed that the expression of cyclin 2 was found in A7r5 cells, and (2) IL-8-induced migration of A7r5 cells showed that with the increase of IL-8 concentration, the number of cell migration increased and the concentration of IL-8 was 20ng/ ml, and the number of cell migration in 50ng/ ml group was higher than that in other groups. Statistical significance, but no difference between the two groups Statistical significance. With the increase of G31P concentration, the number of migrated cells did not change significantly, and there was no difference among the groups. Statistical significance. (4) The results of scratch and migration show that the mean migration distance and the relative migration amount are two statistical values, and only the negative control group treated with G31P is compared with the blank control group. Statistical significance. Compared with blank control group, only the positive control group induced by IL-8 had statistical significance (P0.01). G31P inhibition group was compared with blank control group and G31P negative control group, and the change of two statistical values was not statistically significant. The change in the two statistical values was statistically significant compared to the sexual control (p 0. 01). (5) The results of immunofluorescence assay showed that with the increase of IL-8 concentration, the extension of pseudopodia of the cells increased significantly, and the results showed that, as the concentration of IL-8 increased, In comparison with the cells which were not pretreated with G31P, the final concentration of IL-8 was 20ng/ ml, and after pretreatment with G31P, IL-8 was used to stimulate the cells. The results showed that the fluorescence intensity of calcium ions increased gradually with the increase of IL-8 concentration (0-100ng/ ml). Statistical significance. Compared with the IL-8 stimulation group with the same concentration, the fluorescence intensity of calcium ions in the cells weakened by the stimulation of IL-8, and the fluorescence intensity of intracellular calcium ions weakened. Conclusion: (1) IL-8 can induce the migration of A7r5 cells, the extension of cells and the increase of intracellular calcium ion concentration. It is dose-dependent. (2) G31P can effectively inhibit IL-8 to A7r. Induction of 5 cells. (3) The induction of IL-8 on A7r5 cells may be mediated by the regulation of calcium ion channels in cell membranes
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
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