褪黑素对小鼠肝癌细胞增殖及p27Kip1、cyclin D1基因表达的影响

目的探讨褪黑素(melatonin MT)抑制H22小鼠肝癌细胞增殖的可能机制.方法通过体外细胞培养,分别采用不同剂量MT作用不同的时间,MTT显色技术检测细胞的增殖;RT-PCR检测MT作用4 d后,H22细胞中p27Kip1、cyclin D1mRNA的表达;免疫组化检测H22细胞中p27Kip1、cyclin D1蛋白的表达.结果MT具有抑制肝癌细胞的生长和增殖的作用,且具有时间依赖关系.MT诱导细胞凋亡的过程中,与对照组相比,MT药理剂量组H22细胞中的p27Kip1 mRNA及蛋白表达升高(P＜0.01);cyclin D1 mRNA及蛋白表达下降(P＜0.01).MT生理剂量组p27Kip1mRNA表达升高(P＜0.01),而p27Kip1蛋白表达水平与对照组相比差异无显著性(P＞0.05),cyclinD1 mRNA及蛋白表达均下降,差异元显著性(P＞0.05).结论MT抑制肝癌细胞的增殖可能与上调细胞周期抑制因子p27Kip1的表达,降低周期蛋白cyclin D1表达,进而延迟细胞周期的进程有关.

Effect of melatonin (MT) on proliferation of H22 cells and expression of p27Kip1 and cyclin D1 genes

Objective To study the effect of MT on the growth of mouse hepatocarcinoma cell line H22, and the role of p27Kip1and cyclin D1 in this effect. Methods H22 cells were treated with medical dose (10-6 mol/L) and physiological dose(10-9mol/L) of MT for ld.2d,3d, and 4d, then the cell proliferations were detected by MTT method. RT-PCR and immunohistochemical staining were used to detect the mRNA as well as protein level of p27Kip1 and cyclin Dl in H22 cells that had been treated with MT for four days. Results MT could inhibit the proliferation of H22, it was time dependent. Compared with those in control group, the protein and mRNA levels of p27Kip1 in MTm (medical dose) group were significantly up-regulated, while the expression of cyclin Dl in MTm group was significantly decreased. However, there was no significant difference in the expression of cyclin Dl in protein and mRNA between the control and MTp (physiological dose)group. Conclusion MT could inhibit H22 cell proliferation by up-regulating expression of p27Kip1 and reducing the expression of cyclin D1.