葛根总黄酮体外诱导SHI-1细胞凋亡的分子机制  

Molecular Mechanism of SHI-1 Cell Apoptosis Induced by Puerariae Radix Flavones in Vitro

作　　者：朱国华[1,2] 张琦[1] 戴海萍[3] 季鸥[1] 沈群[2] 

机构地区：[1]南京中医药大学西医诊断学教研室,江苏南京210046 [2]南京中医药大学第一附属医院血液科,江苏南京210029 [3]苏州大学第一附属医院血液科,江苏苏州215006

出　　处：《中国实验血液学杂志》2013年第6期1423-1428,共6页Journal of Experimental Hematology

基　　金：国家自然科学基金(编号81274139);江苏省2012年度普通高校研究生科研创新计划项目(编号CXLX12-0583)

摘　　要：本研究旨在探讨葛根总黄酮(puerariae radix flavoues,PRF)对人急性单核细胞白血病细胞株SHI-1凋亡的影响及其可能分子机制.以不同浓度PRF在不同时间作用于SHI-1细胞,MTT检测细胞增殖抑制率,流式细胞术检测细胞周期,实时定量PCR检测Caspase-3、Caspase-8、Caspase-9、Bcl-2及MLL-AF6融合基因mRNA的表达,Western blot检测MAPK、p-MAPK及NF-κB凋亡相关通路蛋白的表达,明胶酶谱法检测细胞培养上清中MMP的活性。结果表明,PRF呈时间-剂量依赖性抑制SHI-1细胞增殖,使细胞阻滞于S期。以25、50及75μg/ml的PRF分别处理SHI-1细胞,Caspase-3、Caspase-8及Caspase-9在mRNA水平呈浓度依赖性上升(P<0.05),Bcl-2呈浓度依赖性下降但差异无统计学意义(P>0.05),而融合基因MLL-AF6的mRNA与对照组相比无明显变化。将不同浓度PRF作用于SHI-1细胞,胞内JNK、p-JNK、P38 MAPK及p-P38 MAPK的表达均呈浓度依赖性上升(P<0.01);pERK1/2及NF-κB的表达则呈浓度依赖性下降(P<0.01);另细胞培养上清中MMP-2及MMP-9的活性在各处理组中基本不变。结论:一定浓度PRF可体外诱导SHI-1细胞的凋亡,具体分子机制可能与活化Caspases水解酶、激活MAPK、下调NF-κB及Bcl-2等信号分子有关,而与MLL-AF6融合基因无明显相关性。This study was purposed to explore the effects induced by puerariae radix flavones （PRF） on human acute myeloid leukemia SHI-1 cells, apoptosis induced by PRF in vitro and its molecular mechanism. SHI-1 cells were treated with PRF in various concemation, then the inhibitory effect of cell proliferation were detected by MTT method, the cell cycle was analyzed by flow cytometry, the mRNA expression levels of Caspase-3, Caspase-8, Caspase-9, Bcl-2 and MLL-AF6 were detected by real-time polymerase chain reaction （R-T PCR）, the protein expression levels of MAPK, p- MAPK and NF-κB were assayed by Western blot, and the activity of MMP was analyzed by Gelatin zymography. The results indicated that the PRF could inhibit the proliferation of SHI-1 cells in a time-and dose-dependent manner, and the cell cycle was arrested in S phase. When SHI-1 ceils were treated with 25, 50 and 75 μg/ml PRF respectively, mRNA levels of Caspase-3, Caspase-8 and Caspase-9 increased in a dose-dependent manner （P 〈 0.05）, Bcl-2 mRNA decreasd in a concentration-dependent manner （ P 〉 0. 05 ）, and the mRNA level of fusion gene MLL-AF6 did not changed as compared with the control group. Different concentration of PRF was used to treat SHI-1 cells, the expression levels of intracellular JNK, p-JNK, P38 MAPK and p-P38 MAPK increased in the concentration-dependent manner （ P 〈 0. 01 ） ; the expression of p-ERK1/2 and NF-κB decreased in the concentration-dependent manner, and the activity of MMP-2 and MMP-9 in the cell supernatant did not change in each groups. It is concluded that a certain concentration of PRF can induce the apoptosis of SHI-1 cells in vitro, its molecular mechanism may be related to the activation of Caspase hydrolase, activation of MAPK, downregulation of NF-κB, Bcl-2 and other signal molecules. However, it seemed that all these effects are not relate with the MLL-AF6 fusion gene.

关 键 词：葛根总黄酮 SHI-1细胞 细胞凋亡 分子机制 

分 类 号：R733.7[医药卫生—肿瘤] R979.1[医药卫生—临床医学]