瘦素通过调控细胞周期蛋白促进人肝癌细胞增殖

Leptin facilitates the proliferation activity of hepatoma cells via regulating cell cycle proteins

目的:观察瘦素对人肝癌细胞株Huh 7增殖活性的影响,并初步探讨其可能的机制。方法:在培养液中加入不同浓度的瘦素后,采用四甲基偶氮唑盐法(MTT)检测细胞增殖,流式细胞仪检测细胞周期,实时荧光定量PCR和免疫细胞化学法检测细胞周期调控蛋白Cyclin D1和P21waf1的表达。结果:MTT检测显示,瘦素可促进Huh 7细胞的增殖,有一定的时间和剂量依赖性;流式细胞仪检测结果显示,瘦素能明显降低G0/G1期细胞比例并提高S期细胞比例;瘦素(100 ng/ml)处理24 h后,实时荧光定量PCR检测发现Cyclin D1 mRNA表达量增高至对照组的3.15倍,P21waf1mRNA表达量则为对照组的55%,两者均有显著性差异(P<0.01)。免疫细胞化学染色结合图像分析系统分析也发现100 ng/ml的瘦素处理24 h后,与对照组相比,Cyclin D1蛋白的表达明显增高(P<0.01),P21waf1蛋白的表达明显降低(P<0.01)。结论:瘦素可通过促进Cyclin D1表达、抑制P21waf1表达,使人肝癌细胞Huh7由G0/G1期向S期转换,从而促进其增殖。

Objective：To explore the effect of leptin on proliferation of human hepatocellular carcinoma cells Huh 7 and its probable molecular mechanism. Methods：The human hepatocellular carcinoma cells Huh 7 cultured in vitro was treated with leptin at different concentrations. The proliferation of the cells was measured by MTT assay. The cell cycle was monitored by flow cytometry analysis （FCM）. The expression of cell cycle regulatory proteins Cyclin D1 and P21 w,n were determined by immunocytochemistry and imageanalysis system, and real-time quantitative PCR. Results：Leptin significantly raised the proliferation rate of Huh 7 cells. The effect was dose and time-depended partly. Leptin promoted Huh 7 cells in entering the S phase from the G0/G1 phase, mRNA of Cyclin D1 in the leptin treated Huh 7 cells was increased, as compared with that in the control cells （P 〈0.01）. In contrast, P21^waf1 mRNA in the leptin treated Huh 7 cells was decreased as compared with that in the control （P 〈 0.01 ）. Cyclin D1 protein expression in the Huh 7 cells treated with 100 ng/ml leptin was also significantly increased compared with the controls （P 〈 0.01） while the P21^waf1 protein expression was suppressed （P〈0.01）. Conclusion： Leptin can stimulate Cyclin D1 expression and inhibit P21^waf1 expression, promote Huh 7cells entering the S phase from the G0/G1 phase, and facilitate cell proliferation.