IL-8介導(dǎo)血管平滑肌細(xì)胞遷移機(jī)制及G31P的作用初探
發(fā)布時(shí)間：2018-10-31 07:14
【摘要】：目的:動(dòng)脈粥樣硬化(Atherosclerosis, AS)導(dǎo)致的心腦血管疾病是人類健康的最大殺手,在我國(guó)的發(fā)病率逐年升高。近年來(lái)的研究表明AS是一種炎癥性疾病,大量的炎癥細(xì)胞和炎性因子伴隨在其發(fā)生和發(fā)展過(guò)程的始終。炎癥細(xì)胞參與AS表現(xiàn)為在損傷部位有大量炎癥細(xì)胞的粘附、浸潤(rùn),并且釋放大量的炎性因子,趨化白細(xì)胞穿過(guò)血管內(nèi)膜,誘導(dǎo)平滑肌細(xì)胞表型改變,繼而遷移進(jìn)入內(nèi)膜并進(jìn)行增殖。這些細(xì)胞進(jìn)入內(nèi)膜后,開(kāi)始吞噬脂質(zhì)形成泡沫細(xì)胞,加速AS的發(fā)展。趨化因子是一類具有趨化作用的小分子量分泌型蛋白,可以誘導(dǎo)白細(xì)胞進(jìn)入血管內(nèi)膜,在AS中發(fā)揮重要作用。白細(xì)胞介素-8(Interleukin-8, IL-8)是一種單核/巨噬細(xì)胞分泌的多肽,通過(guò)與其受體CXCR1/CXCR2結(jié)合從而啟動(dòng)中性粒細(xì)胞趨化反應(yīng)。IL-8還能夠誘導(dǎo)人主動(dòng)脈血管平滑肌細(xì)胞遷移和增殖;并且可以促進(jìn)AS中新生血管形成,但其具體機(jī)制尚不清楚。抑制IL-8與其受體結(jié)合為治療AS提供了新的思路。G31P是通過(guò)對(duì)人IL-8基因的定點(diǎn)突變,篩選的出高親和力,無(wú)活性的人IL-8類似物。通過(guò)實(shí)驗(yàn)證實(shí)了G31P可以高效的親和CXCR1/CXCR2,對(duì)與中性粒細(xì)胞相關(guān)的感染治療上有明顯的療效。G31P對(duì)中性粒細(xì)胞CXCR1/CXCR2的有效抑制作用能否發(fā)生在血管平滑肌細(xì)胞中尚未有研究。 方法:(1)利用RT-PCR的方法檢測(cè)A7r5細(xì)胞中是否有IL-8的受體之一CXCR2的表達(dá);(2)利用Boyden Chamber法觀察IL-8和G31P對(duì)A7r5細(xì)胞遷移的影響;(3)利用劃痕法和Boyden Chamber法觀察G31P在IL-8誘導(dǎo)A7r5細(xì)胞遷移過(guò)程中的影響;(4)利用細(xì)胞免疫熒光方法觀察細(xì)胞骨架變化情況;(5)利用鈣離子熒光探針Fluo-3 AM檢測(cè)IL-8與G31P對(duì)A7r5細(xì)胞內(nèi)鈣離子濃度的影響。 結(jié)果:(1)RT-PCR結(jié)果顯示A7r5細(xì)胞中有CXCR2的表達(dá);(2)IL-8誘導(dǎo)A7r5細(xì)胞遷移實(shí)驗(yàn)結(jié)果顯示,隨著IL-8濃度的增加,細(xì)胞遷移數(shù)呈現(xiàn)上升趨勢(shì),IL-8濃度為20ng/ml、50ng/ml兩組的細(xì)胞遷移數(shù)較其它各組有統(tǒng)計(jì)學(xué)意義,但這兩組之間差異無(wú)統(tǒng)計(jì)學(xué)意義。隨著G31P濃度的增加,遷移的細(xì)胞數(shù)并未發(fā)生明顯變化,各組間的差異無(wú)統(tǒng)計(jì)學(xué)意義。(4)劃痕和遷移實(shí)驗(yàn)結(jié)果顯示了細(xì)胞平均遷移距離與相對(duì)遷移數(shù)量?jī)身?xiàng)統(tǒng)計(jì)值的變化情況,只用G31P處理的陰性對(duì)照組與空白對(duì)照組比較,兩項(xiàng)統(tǒng)計(jì)值的變化均無(wú)統(tǒng)計(jì)學(xué)意義。只用IL-8誘導(dǎo)的陽(yáng)性對(duì)照組與空白對(duì)照組比較,兩項(xiàng)統(tǒng)計(jì)值的變化均有統(tǒng)計(jì)學(xué)意義(p0.01)。G31P抑制組與空白對(duì)照和G31P陰性對(duì)照組比較,兩項(xiàng)統(tǒng)計(jì)值的變化無(wú)統(tǒng)計(jì)學(xué)意義;與IL-8陽(yáng)性對(duì)照比較,兩項(xiàng)統(tǒng)計(jì)值的變化有統(tǒng)計(jì)學(xué)意義(p0.01)。(5)免疫熒光實(shí)驗(yàn)結(jié)果顯示,隨著IL-8濃度增加,細(xì)胞偽足伸出明顯增加,呈劑量依賴趨勢(shì)。與未用G31P預(yù)處理、IL-8終濃度為20ng/ml組的細(xì)胞相比,在用G31P預(yù)處理后再用IL-8刺激,可觀察到細(xì)胞偽足的伸出不明顯。(6)細(xì)胞內(nèi)鈣離子濃度測(cè)定結(jié)果顯示,隨著IL-8濃度的增加(0-100ng/ml)細(xì)胞內(nèi)鈣離子的熒光強(qiáng)度逐漸增加,呈現(xiàn)劑量依賴趨勢(shì),與對(duì)照組相比有統(tǒng)計(jì)學(xué)意義。與用同樣濃度的IL-8刺激組相比,用G31P預(yù)處理的細(xì)胞、再用IL-8刺激后,其細(xì)胞內(nèi)鈣離子的熒光強(qiáng)度變?nèi)?有統(tǒng)計(jì)學(xué)意義。 結(jié)論:(1)IL-8可以誘導(dǎo)A7r5細(xì)胞的遷移、細(xì)胞偽足伸出、細(xì)胞內(nèi)鈣離子濃度升高,具有劑量依賴性。(2)G31P可以有效抑制IL-8對(duì)A7r5細(xì)胞的誘導(dǎo)作用。(3)IL-8對(duì)A7r5細(xì)胞的誘導(dǎo)作用可能通過(guò)調(diào)節(jié)細(xì)胞膜鈣離子通道進(jìn)而調(diào)節(jié)細(xì)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
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：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
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363
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