甘草对小肠隐窝干细胞增殖中p53mRNA稳定性及其表达的影响

Effects of Licorice on p53 mRNA Stability and Expression during the Proliferation of Intestinal Crypt Stem Cells

目的研究甘草对小肠隐窝干细胞IEC-6细胞增殖及p53表达的影响。方法采用二氟甲基鸟氨酸(DFMO)耗竭细胞内多胺的方法诱导IEC-6细胞生长抑制模型,实验分4组,即对照组、DFMO组、甘草低剂量组和甘草高剂量组。对照组为正常细胞,其他3组均加入5 mmol/L DFMO,同时甘草低剂量组与甘草高剂量组分别添加40、80μg/mL甘草配方颗粒,均连续培养6天。利用流式细胞术检测各组细胞数量及存活率,Western blot检测p53蛋白水平,荧光定量RT-PCR检测p53 mRNA水平及mRNA稳定性。结果与对照组比较,第4天DFMO组IEC-6细胞生长明显受到抑制(P<0.05);第6天甘草低剂量组及甘草高剂量组细胞数量较DFMO组明显增加(P<0.05),且2个剂量的甘草组间具有量效依赖性。处理IEC-6细胞6天后,DFMO组p53蛋白及mRNA表达水平较对照组显著升高(P<0.05);与DFMO组比较,2个剂量的甘草组能够明显下调p53蛋白及mRNA的表达水平(P<0.05)。对照组细胞的p53 mRNA降解最快,DFMO组细胞的降解速度最慢。结论甘草通过下调p53 mRNA的稳定性来降低p53表达,从而促进小肠隐窝干细胞的增殖,可能是甘草的肠黏膜保护和修复作用的机制之一。

Objective To study the effects of licorice on the proliferation of intestinal crypt stem cell line IEC-6 and the expression of p53.Methods Induced by difluoro-methylornithine（DFMO）,polyamine-depleted IEC-6 cells under growth inhibition were used as the pathological cell model in this study.Cells were divided into four groups,i.e.,the control group,the DFMO-treated group,the high dose licorice group,and the low dose licorice group.The control group consisted of IEC-6 cells cultured in normal condition.The other three groups were all treated with 5 mmol/L DFMO.The high dose and low dose licorice groups were supplemented with 40 and 80 μg/mL licorice granule respectively.All the groups were cultured for 6 successive days.The cell number and viability were determined using flow cytometry.The level of p53 protein was detected by Western blot.The p53 mRNA levels and stability were detected using fluorescent quantitative Real-time PCR.Results Compared with the control group,the cell growth of the DFMO group was obviously inhibited on the 4th day（P0.05）.The cell number increased more obviously in the low dose licorice and the high dose licorice groups in a dose-dependent way on the 6th day when compared with the DFMO group（P0.05）.When compared with the control group,significantly elevated expression levels of p53 protein and mRNA in cells of the DFMO group were detected after 6-day treatment（P0.05）.When compared with the DFMO group,the expression levels of p53 protein and mRNA were significantly down-regulated in the low dose licorice and the high dose licorice groups（P0.05）.The degradation of p53 mRNA was the fastest in the control group,while the degradation speed of cells in the DFMO group was the slowest.Conclusion One of mechanisms for protective and healing effects of licorice on the intestinal mucosa was possibly through down-regulating the stability of p53 mRNA,lowering the expression of p53,thus promoting the proliferation of the intestinal crypt stem cells.