蛋氨酸脑啡肽对神经母细胞瘤细胞生长周期的影响及其机制研究

Effect of methionine enkephalin on growth cycle of neuroblastoma cell and its mechanism research

目的:研究蛋氨酸脑啡肽(Met-ENK)对神经母细胞瘤细胞SH-SY5Y的生长抑制作用及细胞周期的抑制机制。方法:采用MTT法检测细胞生长抑制率;流式细胞术检测Met-ENK作用后肿瘤细胞周期变化;Real-time PCR法检测Met-ENK作用后细胞内P21,P53基因转录量变化;Western blot法检测细胞内P21,P16,磷酸Rb蛋白表达量的变化情况。结果:MTT法结果显示,Met-ENK对SH-SY5Y细胞有生长抑制作用,在Met-ENK浓度为10-5mol·L-1时抑制率为40%;流式细胞术证实Met-ENK作用后大多数细胞被抑制于细胞周期的G0/G1期,该期细胞数由63.97%变为87.73%;P21的基因转录量是用药前的3倍,P53基因转录量是用药前的5倍;P21,P16蛋白表达量明显增加,磷酸Rb蛋白表达量明显降低。结论:Met-ENK可以通过上调细胞周期蛋白依靠性激酶抑制因子P21及抑癌基因P53的转录和蛋白表达量,下调Rb蛋白的磷酸化水平将SHSY5Y细胞的增殖抑制于生长周期的G0/G1期,表明Met-ENK的G0/G1期特异性周期抑瘤作用可以通过调节该途径产生。

Objective： To study the cell growth inhibition and cell cycle inhibitory mechanisms of methionine enkephalin （Met-ENK） on SH-SY5Y cell. Methods： MTT assay was used for detecting the cell growth inhibition rate, the change of tumor cell cycle was detected by flow cytometry, the levels of intracellular P21 and P53 gene transcript were analyzed by Real-time PCR, the expressions of intracellular P21, P16 and phosphorylated Rb protein were determined using Western blot assay after intervention with Met-ENK. Results： MTT assay showed that Met-ENK could inhibit SH-SY5Y cell growth, and the inhibition rate was 40% at the Met-ENK concentration of 10-2 mol.Ll. The results of flow cytometry confirmed that most of cells were blocked in G0/G1 phase, and the number of cells in G0/G1 phase increased from 63.97% to 87.73%. The transcript levels of P21 gene and P53 gene were three and five times higher than that before intervention with Met-ENK. The expressions of P21 and P16 protein significantly increased, while expression of phosphorylation Rb protein significantly reduced. Conclusion： Met-ENK could inhibit the SH-SY5Y cell proliferation in the G0/G1 phase of the growth cycle by increasing the transcription and protein expression of the cyclin-dependent kinase inhibitor-P21 and tumor suppressor gene-P53 and decreasing the level of Rb protein phosphorylation, which confirmed the mechanism of G0/G 1 phase specificity cycle inhibitory effect of Met-ENK.