维甲酸对异位内膜基质细胞Beclin-1的影响研究

Effect of retinoic acid on Beclin-1 of endometriotic stromal cells

目的探讨不同剂量维甲酸(RA)对正常子宫内膜基质细胞和异位内膜基质细胞(ESC)Beclin-1的影响。方法体外培养正常子宫内膜基质细胞和ESC,分为空白对照组(正常子宫内膜基质细胞),氯喹(CQ)组(ESC),CQ+RA组(ESC)。分别采用RT-PCR及Western blot检测各组LC3-Ⅱ、Beclin-1mRNA及蛋白的表达量,PCR及Western blot检测RA作用于CQ或转染siRNA Beclin-1处理的细胞中Beclin-1表达的变化,MTT法检测RA细胞毒性。结果 CQ组LC3-Ⅱ、Beclin-1mRNA表达较空白对照组减少,CQ+RA组较CQ组增加,差异有统计学意义(P<0.05);CQ+RA组与空白对照组LC3-ⅡmRNA表达比较差异无统计学意义(P>0.05)。随着RA浓度的增加,ESC Beclin-1表达逐渐升高,差异有统计学意义(P<0.05)。siRNA Beclin-1在确定的转染条件下可有效沉默ESC的Beclin-1基因表达,RA可解除siRNA对Beclin-1的沉默作用。不同浓度RA对细胞的存活率与对照组比较,差异无统计学差异(P>0.05)。结论 RA可以诱导ESC中自噬的发生,升高ESC Beclin-1的表达,且与刺激浓度呈正相关。

Objective To detect the effect of change of different doses of retinoic acid(RA)on Beclin-1 expression of normal endometrial stromal cells and endometriotic stromal cells(ESC),and to preliminarily discuss the action mechanism of RA in treating endometriosis.Methods In vitro cultured normal endometrial stromal cells and ESC were divide into the blank control group(normal endometrial stromal cells),chloroquine(CQ)group(ESC cells)and CQ and RA combined action group(ESC cells).RT-PCR and Western blot were adopted to detect LC3-Ⅱand Beclin-1 mRNA and protein expression levels in each group;PCR and Western blot were used to test Beclin-1 expression level in cells by RA acting on CQ or by transfecting siRNA treatment;MTT was used to detect cytotoxicity of RA.The inter-group comparison adopted one-way analysis of variance,and the inter-group pairwise comparison adopted the t test.Results The expression of LC3-ⅡmRNA and Beclin-1 mRNA in CQ+RA group was lower than that in blank control group,and the expression of LC3-ⅡmRNA in CQ+RA group was higher than that in CQ group(P<0.05).With the increase of RA concentration,the expression of ESC Beclin-1 increased gradually,and the difference was statistically significant(P<0.05).SiRNA Beclin-1 could effectively silence the Beclin-1 gene expression of ESC under certain transfection conditions,and RA could relieve the silencing effect of siRNA.There was no significant difference in survival rate between RA group and control group at different concentrations(P>0.05).Conclusion RA could induce the occurrence of autophagy in ESC,enhances Beclin-1 expression in ESC and is positively correlated with the stimulation concentration.