外源性视黄酸诱导乳鼠心肌细胞凋亡的研究

发布时间：2018-06-01 21:13

  本文选题：视黄酸 + 心肌细胞　； 参考：《桂林医学院》2012年硕士论文

【摘要】：目的：研究外源性视黄酸(Retinoic acid, RA)对乳鼠心肌细胞凋亡的影响,并研究乳鼠心肌细胞中Fas,Fas1, Casepase-3,Pax-8细胞调控因子的表达水平,分析RA诱导心肌损伤的机制。方法：1.体外培养乳鼠原代心肌细胞以及明确心肌细胞干预的最佳时间：在无菌条件下取出Balb/c乳鼠心脏,采用胰酶-差速贴壁法分离并纯化获得乳鼠心肌细胞,并进行原代心肌细胞培养。2.RA干预心肌细胞后进行指标的检测：设置的空白对照组为选用含20%胎牛血清的DMEM高糖培养基培养心肌细胞；实验组分别使用含不同浓度视黄酸的培养基干预心肌细胞,在用药干预后的0h、12h、24h、48h使用倒置显微镜观察心肌细胞形态并动态记录细胞搏动力；用MTT法来检测心肌细胞的抑制率；流式细胞仪检测心肌细胞凋亡率；3.RA干预心肌细胞后Fas、Fas1、Caspase-3、Pax-8调控因子检测：Western blot测定Fas、Fas1、Caspase-3、Pax-8蛋白的表达水平。RT-PCR检测Fas、Fasl mRNA表达水平。结果：1.成功培养Balb/c乳鼠心肌原代细胞。培养72h的心肌细胞生长旺盛,表现为搏动力强,心肌细胞增殖逐渐进入平台期,为最佳的细胞干预期。2.视黄酸浓度≥2umo1/L时能显著抑制正常Balb/c乳鼠心肌细胞的生长,使细胞的搏动力减弱、凋亡增加,同时干预的药物浓度越高、时间越长,抑制越明显。3.与对照组相比,2umol/L RA组随作用时间的延长,Fas、Fas1、Caspase-3蛋白的表达逐渐增强(P0.05)；与之相对应的是Pax-8蛋白的表达则逐渐降低(P0.05)；Fas、Fas1mRNA表达逐渐增强(P0.05)。结论：1.采用胰酶-差速贴壁法及化学药物抑制法能获取存活率高、纯度高的心肌原代细胞；心肌细胞培养72h为最佳的心肌细胞干预时间点。2.高浓度RA能抑制心肌细胞生长,使细胞搏动力减弱或消失,并诱导心肌细胞凋亡。3.RA通过调节Fas、Fas1、Caspase-3、Pax-8的表达,抑制正常心肌细胞的增殖,促进细胞的凋亡。
[Abstract]:Aim: to study the effect of Retinoic acid (RA) on cardiomyocyte apoptosis and the expression of Fas-Fas1, Casepase-3 Pax-8 in neonatal rat cardiomyocytes, and to analyze the mechanism of myocardial injury induced by RA. Method 1: 1. The optimal time for primary culture of neonatal rat cardiomyocytes in vitro and the intervention of cardiomyocytes were as follows: the hearts of Balb/c rats were removed under aseptic conditions and isolated and purified by trypsin differential adherent method. The primary cardiomyocyte culture. 2. The indexes were detected after RA intervention. The blank control group was cultured on DMEM medium containing 20% fetal bovine serum. The experimental groups were treated with different concentrations of retinoic acid to intervene cardiomyocytes. The morphology of cardiomyocytes was observed and the pulsatility of cardiomyocytes was recorded dynamically by inverted microscope at 0 h, 12 h, 24 h and 48 h after intervention, and the inhibition rate of cardiomyocytes was detected by MTT method. Apoptosis rate of cardiomyocytes was measured by flow cytometry. After RA intervention, the expression of Fas-Fas1Caspase-3 and Pax-8 was detected by Western blot. The expression level of Fas-Fas1Caspase-3 and Pax-8 protein was detected by RT-PCR. The expression of Fas-Fasl mRNA in Fas-Fas1Caspase-3 was detected by reverse transcription-polymerase chain reaction (RT-PCR). The result is 1: 1. Primary myocardial cells were successfully cultured in neonatal Balb/c mice. After 72 hours of culture, cardiomyocytes grew vigorously, showing strong pulsatility, and the proliferation of cardiomyocytes gradually entered the plateau phase, which was the best cell dry expectation. 2. When the concentration of retinoic acid 鈮，

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