摘要
目的研究羧甲基茯苓多糖(carboxymethylpachymaran,CMP)对前列腺癌细胞周期的影响。方法利用四甲基偶氮唑盐[3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide,MTT]检测筛选出1、10、50、100、250、500、1000μg/ml的CMP对PC-3细胞作用的IC50值,并选出最佳抑制的作用时间点及所对应的CMP浓度。将PC-3细胞随机分为5组:对照组,低、中、高剂量组,阳性对照组。采用Hoechst 33258染色检测细胞形态变化,流式细胞术检测CMP对前列腺癌细胞周期的影响,Western blot检测各组细胞周期相关蛋白Cyclin A、Cyclin D1、CDK2的表达,反转录-聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)检测Cyclin A、Cyclin D1、CDK2 mRNA的表达水平。结果利用MTT法检测不同浓度CMP对PC-3细胞的抑制作用后,选择48 h为后续实验的作用时间点,108.7μg/ml作为后续实验中剂量组的干预剂量。Hoechst 33258染色显示,对照组胞核呈椭圆形,呈淡蓝色,凋亡细胞较少,而其余各组出现胞核缩小,染色质边集,呈亮蓝色。与对照组相比,低、中、高剂量组及阳性对照组细胞的G0/G1期细胞增多,G2/M期细胞减少,Cyclin A、Cyclin D1、CDK2的表达水平降低(P<0.01)。结论CMP对前列腺癌细胞周期具有抑制作用,其机制可能与抑制细胞周期相关蛋白Cyclin A、Cyclin D1、CDK2的表达有关。
Objective To study the effect of carboxymethylpachymaran(CMP)on prostate cancer cell cycle.Methods The IC50 values of 1,10,50,100,250,500,1000μg/ml CMP on PC-3 cells were screened by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide(MTT)assay,and the optimal inhibition time point and corresponding CMP concentration were selected.The PC-3 cells were randomly divided into five groups:control group,low-,medium-and high-dose group,and positive control group.The change of cell morphological was detected by Hoechst 33258 staining,the effect of CMP on cell cycle of prostate cancer cell was detected by flow cytometry,the expression of cell cycle-related proteins Cyclin A,Cyclin D1,CDK2 were detected by Western blot,the mRNA expression of Cyclin A,Cyclin D1,CDK 2 were detected reverse transcription polymerase chain reaction(RT-PCR).Results After using MTT assay to detect the inhibitory effect of different concentrations of CMP on PC-3 cells,48 h was selected as the time point of the subsequent experiment,and 108.7μg/ml was used as the intervention dose of the medium-dose group in the subsequent experiment.Hoechst 33258 staining showed that nucleus in the control group was oval,evenly stained,pale blue and less apoptosis cells.The nucleus of the other groups was shrinking,nuclear chromatin edge set,showing a bright blue color.Compared with the control group,G0/G1 phase cells were increased and G2/M phase cells were decreased,the expression levels of Cyclin A,Cyclin D1,CDK2 were significantly decreased of the low-,medium-and high-dose groups and the positive control group(P<0.01).Conclusion CMP can inhibit the cell cycle of prostate cancer cells,and its mechanism may be related to the inhibition of the expression of Cyclin A,Cyclin D1 and CDK2.
作者
汪芳
陈琪
吴颖涛
倪文旭
WANG Fang;CHEN Qi;WU Yingtao;NI Wenxu(Department of Laboratory,General Hospital of Central Theater Command,Wuhan Hubei 430070,China)
出处
《华南国防医学杂志》
CAS
2020年第11期762-767,共6页
Military Medical Journal of South China
