雌激素对乳腺癌干细胞增殖、自我更新的作用研究

Effects of Estrogen on Proliferation and Self-Renewal of Breast Cancer Stem Cells

目的探讨雌激素对乳腺癌干细胞(BCSC)增殖及自我更新的影响。方法以人乳腺癌细胞MCF-7培养BCSC,并采用细胞免疫荧光实验验证;用含0,1,5,10,20,100 nmol/L雌激素的完全培养基培养BCSC 0,24,48,72 h后,采用CCK-8法检测细胞增殖情况;用含0,10,20 nmol/L雌激素的完全培养基培养BCSC 24 h后,观察细胞形态并计算成球率;用含0,1,5,10 nmol/L雌激素的完全培养基培养BCSC 24 h后,采用荧光定量聚合酶链式反应(PCR)法检测磷脂酰肌醇3激酶(PI3K)、蛋白激酶B(AKT)和哺乳动物雷帕霉素靶蛋白(mTOR)mRNA的表达情况。结果成功从MCF-7细胞中培养出BCSC。与0 nmol/L比较,1,5,10 nmol/L雌激素作用下,BCSC增殖能力显著增强(P<0.05),且与浓度和作用时间呈正相关;与0 nmol/L比较,10 nmol/L和20 nmol/L雌激素作用下,BCSC体积均显著增大,数量均显著增多(P<0.05);与0 nmol/L比较,1,5,10 nmol/L雌激素作用下,BCSC的PI3K,AKT,mTORmRNA表达水平均显著升高(P<0.05),且存在浓度依赖性。结论较低浓度(0~10 nmol/L)的雌激素具有促进BCSC生长的作用,其作用机制可能与活化PI3K/AKT/mTOR信号通路有关。

Objective To investigate the effects of estrogen on the proliferation and self-renewal of breast cancer stem cells(BCSCs).Methods BCSCs were cultured from human breast cancer cells MCF-7,and they were verified by the cell immunofluorescence test.After the breast BCSCs were cultured in complete medium containing 0,1,5,10,20,100 nmol/L estrogen for 0,24,48,72 h,the cell proliferation was detected by the CCK-8 method.After the BCSCs were cultured in complete medium containing 0,10,20 nmol/L estrogen for 24 h,the cell morphology was observed and the spheroidization rate was calculated.After the BCSCs were cultured in complete medium containing 0,1,5,10 nmol/L estrogen for 24 h,the expression of phosphatidylinositol 3-kinase(PI3K),protein kinase B(AKT)and mammalian target of rapamycin(mTOR)mRNA was detected by the fluorescence quantitative polymerase chain reaction(PCR).Results BCSCs were successfully cultured from MCF-7 cells.Compared with that at 0 nmol/L estrogen,the proliferation ability of BCSCs at 1,5,10 nmol/L estrogen significantly increased(P<0.05),and that was positively correlated with the concentration and time.Compared with those at 0 nmol/L estrogen,the volume and quantity of BCSCs at 10 nmol/L and 20 nmol/L estrogen significantly increased(P<0.05).Compared with those at 0 nmol/L estrogen,the expression levels of PI3K,AKT and mTOR mRNA in BCSCs at 1,5,10 nmol/L estrogen significantly increased(P<0.05),with a concentration-dependent manner.Conclusion Low concentration(0-10 nmol/L)of estrogen can promote the growth of BCSCs,and its mechanism may be related to the activation of PI3K/AKT/mTOR signaling pathway.