姜黄素对结肠癌细胞HT-29中干细胞标记物CD24及Lgr5增殖的影响

The effect of curcumin on proliferation of CD24and Lgr5in colon cancer cell lines HT-29

[目的]探讨姜黄素对结肠癌细胞HT-29中干细胞的抑制作用,为结肠癌提供靶向性治疗及理论基础。[方法]采用流式细胞仪分选HT-29中CD24阳性表达细胞;分选出带有荧光标记物CD24的细胞接种于无血清培养基中,培养观察细胞的成球状态;加入10μmol/L、30μmol/L、60μmol/L的姜黄素干预后再次通过流式细胞仪分选HT-29细胞株中CD24阳性细胞表达;采用RT-PCR检测结肠癌干细胞CD24中Lgr5mRNA的表达;利用免疫组化检测加入姜黄素后观察Lgr5蛋白的表达。[结果]通过流式细胞仪分选HT-29细胞株CD24所占比例为40%,将分选出的CD24阳性细胞接种于无血清培养基中培养,10d后成球;加10μmol/L、30μmol/L、60μmol/L的姜黄素干预后再次通过流式细胞仪分选HT-29细胞株CD24所占比例分别为20.23%、13.04%、7.54%;在mRNA表达水平10μmol/L、30μmol/L、60μmol/L姜黄素作用于HT-29细胞,能够使分选出的细胞中CD24和Lgr5mRNA的表达比较呈下降趋势(P<0.05)。通过免疫组化检测,姜黄素干预后,Lgr5随浓度的增加,其表达呈下降趋势(P<0.05)。[结论]姜黄素对肿瘤干细胞具有抑制作用,为姜黄素的抗肿瘤机制提供另一理论途径。

[Objective]To investigate the inhibitory effect of curcumin on tumor stem cells and to provide the base of targeted therapy and theoretical basis for colon cancer. [Methods]The CD24positive expression cells in HT-29were selected by flow cytometry. Then cells with fluorescent marker CD24were incubated in serum-free medium to observe the formation of sphere of stem cells. After the intervention of 10 μmol/L, 30μmol/L and 60 μmol/L, the expression of CD24positive cells in HT-29cell lines were selected by flow cytometry againRt-PCR and were used to detect the expression of Lgr5mRNA in CD24colon cancer stem cells positive. The immunocytochemical method was applied to detect Lgr5protein expression after adding curcumin. [Results] The proportion of CD24 incubated expression in HT-29cell line was 40% by flow cytometry. The CD24positive cells were incubated in the serum-free medium and formed into sphere of cells after lOdays. The proportion of CD24expression in HT-29cell lines was 20. 23 % and 13. 04% ,respectively,withl0μmol/L,30 μmol/L and 60μmol/L curcumin intervention. By immunohistochemistry, the expression of Lgr5 was decreased gradually with the increase of concentration（P〈0. 05）. At the mRNA expression level,10mol/L, 30mol/L,60mol/L curcumin ACTS on HT-29cells,which can reduce the mRNA expression of CD24 and Lgr5 in the selected cells（P〈0. 05）. [Conclusion]Curcumin has inhibitory effect on tumor stem cells and our data provides another theoretical approach for the anti-tumor mechanism of curcumin.