Fractalkine诱导H9C2心肌细胞凋亡及黄芪甲苷的干预作用

发布时间：2019-06-01 18:15

【摘要】：目的探讨可溶性Fractalkine对于H9C2心肌细胞的增殖的影响;进一步探讨可溶性Fractalkine对H9C2心肌细胞凋亡的影响及黄芪甲苷的干预作用。方法首先分别选用 Ong/ml、100ng/ml、200ng/ml、300ng/ml、400ng/ml、500ng/ml的可溶性Fractalkine刺激H9C2心肌细胞,分别于0、12、24、36、48小时采用MTT法检测可溶性Fractalkine对心肌细胞增殖的作用;计算出可溶性Fractalkine作用的最佳时间及最佳作用剂量;然后分别采用Ong/ml(A组)、200ng/ml(B组)、200ng/ml可溶性Fractalkine+50mg/L黄芪甲苷(C组)刺激H9C2心肌细胞,通过荧光染色检测细胞的凋亡情况;并进一步应用流式细胞仪测定细胞凋亡百分数,通过蛋白免疫印迹(western blotting)及实时定量聚合酶链式反应(RT-qPCR)测定可溶性Fractalkine对H9C2心肌细胞凋亡的影响。计量资料采用均数±标准差((?)±s)表示,采用非配对t检验或单因素方差分析,按P0.05的检验水准,具有统计学意义。结果1.低剂量的可溶性的Fractalkine就能够抑制H9C2心肌细胞的增殖;而且此抑制作用随着可溶性Fractalkine浓度的增加及作用时间的延长而增加,各组间有统计学差异;得出最佳作用时间为24小时,半数抑制剂量为277.5ng/ml;2.通过荧光染色发现可溶性Fractalkine可诱导心肌细胞凋亡;应用黄芪甲苷后可见凋亡细胞减少;3.Annexin-FITC检测细胞凋亡率,显示A组、B组、C组凋亡率分别为4.13 ±0.07%,41.64±0.75%,34.07±0.55%;提示可溶性 Fractalkine 可诱导 H9C2 心肌细胞凋亡(P0.001),而黄芪甲苷可以抑制这种作用(P0.01);4.Western blotting结果显示,B组Bcl-2的表达减少,Bax的表达增加(P0.05),应用黄芪甲苷后可抑制这种作用(P0.05);5.RT-qPCR结果显示:B组Bcl-2mRNA的表达量明显减少,而BaxmRNA的表达量明显增加(P0.001),应用黄芪甲苷后可抑制这种作用(P0.01)。结论1.可溶性Fractalkine可抑制H9C2心肌细胞增殖,此作用随着可溶性Fractalkine浓度的增加及作用时间的延长而增大;2.可溶性Fractalkine可诱导H9C2心肌细胞发生凋亡;3.黄芪甲苷可减少可溶性Fractalkine诱导的H9C2心肌细胞凋亡。
[Abstract]:Objective to investigate the effect of soluble Fractalkine on the proliferation of H9C2 cardiomyocytes and the effect of soluble Fractalkine on apoptosis of H9C2 cardiomyocytes and the intervention of astragaloside A. Methods H9C2 cardiomyocytes were stimulated by soluble Fractalkine of Ong/ml,100ng/ml,200ng/ml,300ng/ml,400ng/ml,500ng/ml, and the effect of soluble Fractalkine on cardiomyocyte proliferation was detected by MTT assay at 0, 12, 24, 36 and 48 hours, respectively. The optimum time and dose of soluble Fractalkine were calculated. Then Ong/ml (group A), 200ng/ml (group B) and 200ng/ml soluble Fractalkine 50mg/L astragaloside A (group C) were used to stimulate H9C2 cardiomyocytes, and the apoptosis of H9C2 cardiomyocytes was detected by fluorescence staining. The percentage of apoptosis was measured by flow cytometry, and the effect of soluble Fractalkine on apoptosis of H9C2 cardiomyocytes was measured by Western imprinting (western blotting) and real-time quantitative polymerase chain reaction (RT-qPCR). The measurement data were expressed by mean 卤standard deviation (?) 卤s). Unmatched t test or single factor ANOVA analysis were used to show that it was statistically significant according to the test level of P05. Result 1. Low dose of soluble Fractalkine could inhibit the proliferation of H9C2 cardiomyocytes, and the inhibitory effect increased with the increase of soluble Fractalkine concentration and the prolongation of action time, and there was significant difference among the three groups. The optimum action time was 24 hours, and the half inhibitory dose was 277.5 ng 路ml ~ (- 2). It was found that soluble Fractalkine could induce cardiomyocyte apoptosis by fluorescence staining, and apoptotic cells decreased after astragalosides were used. The apoptosis rate of group A, group B and group C was 4.13 卤0.07%, 41.64 卤0.75% and 34.07 卤0.55%, respectively. It is suggested that soluble Fractalkine can induce apoptosis of H9C2 cardiomyocytes (P0.001), while astragalosides can inhibit this effect (P0.01). The results of 4.Western blotting showed that the expression of Bcl-2 decreased and the expression of Bax increased in group B (P 0.05). Astragaloside A could inhibit this effect (P 0.05). The results of 5.RT-qPCR showed that the expression of Bcl-2mRNA in group B was significantly decreased, while the expression of BaxmRNA was significantly increased (P0.001). Astragaloside A could inhibit this effect (P 0.01). Conclusion 1. Soluble Fractalkine could inhibit the proliferation of H9C2 cardiomyocytes, which increased with the increase of soluble Fractalkine concentration and the prolongation of action time. Soluble Fractalkine could induce apoptosis of H9C2 cardiomyocytes. Astragaloside A can reduce the apoptosis of H9C2 cardiomyocytes induced by soluble Fractalkine.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54

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