摘要
目的
观察姜黄素诱导人白血病细胞株K562凋亡作用及其细胞周期的变化,同时观察其对凋亡相关基因BCL-XL和BAK表达的影响。探讨姜黄素抑制人白血病细胞K562生长并诱导凋亡的机制,指导临床白血病的化学药物治疗。
材料与方法
1细胞培养
以人类白血病细胞株K562为研究对象。将K562细胞置于体积分数为10%的新生胎牛血清的RPMI-1640培养基中,37℃、5%二氧化碳孵箱中培养；取对数生长期细胞,0.4%台盼蓝染色,细胞活性在98%以上,调整细胞密度为1×106个/mL,0.9ml/孔接种于24孔板进行实验。
2实验检测
将对照组和经10umol/L、30umol/L、50umol/L、100umol/L的姜黄素干预的实验组细胞于24h和48h后分别收集起来,用流式细胞仪检测凋亡率和细胞周期的变化,RT-PCR方法检测BCL-XL和BAK的mRNA的表达变化,免疫组化法检测BCL-XL和BAK蛋白的表达。
3统计学处理
采用SPSS13.0统计软件处理,数据以均数±标准差(x±s)表示,经方差齐性检验后采用单因素方差分析,以α=0.05为检验水准。
结果
姜黄素可以对K562细胞有较明显的增殖抑制作用,且呈剂量依赖与时间依赖,流式细胞术结果显示姜黄素浓度在100μM时作用24小时后,凋亡率为(58.53±1.08)%,对照组凋亡率为(1.12±0.20)%,与对照组相比明显增高(P<0.05)；姜黄素浓度在100μM时作用48小时后,凋亡率为(84.70±1.53)%,对照组凋亡率为(1.25±0.19)%,与对照组相比明显增高(P<0.05)；随着姜黄素浓度增加,作用于K562细胞后可以使其倍增时间明显延长,G1期细胞增多,S期细胞减少；RT-PCR结果显示姜黄素能下调K562细胞BCL-XL mRNA的表达,上调BAKmRNA的表达(P<0.05),免疫组化法发现姜黄素可以降低BCL-XL蛋白的表达,升高BAK蛋白的表达(P<0.05)。
结论
1姜黄素可以诱导K562细胞凋亡,且凋亡率呈时间和剂量依赖性。
2姜黄素可以影响K562的细胞周期,主要将其阻滞于G1期。
3随姜黄素浓度的增高,上调了BAK的mRNA和蛋白在K562细胞中的表达,下调了BCL-XL的mRNA和蛋白在K562细胞中的表达,可能和p53的信号转导相关。
Objective
To investigate the mechanism of Curcumin induced apoptosis of human leukemia K562 and its effect on expression of apoptosis gene BCL-XL and BAK.
Methods
1 Cell culture
In our study, human erythroleukemia cell line K562 were used as object the K562 cells were cultured in the RPMI-1640 medium with 10%(volume fraction) new-born calf serum, in 37℃,5% CO2 incubator K562 cells in the logarithmic growth phase, after assessment for cell survival rate over 98% by trypan blue, the cell concentration were adjusted to 1×106/mL,were incubated in 24 holes culture plate by 0.9ml/hole.
2 Experment test
K562 Cells were exposed to 0,10,30,50,100 umol/L of Curcumin for 24h and 48h respectively.Then the apoptosis of cells were analyzed by flow cytometry; The BCL-XL and BAK mRNA expression was quantified by Reverse transcription-polymerase chain reaction(RT-PCR).The protein expression of BCL-XL and BAK was semi-quantitatively analyzed with SP techniques.
3 Statistics
All tested data was showed by mean±standard deviation (x±s).Measurement data was compared with sigle factor analysis of variance. P-value< 0.05 was considered statistically significant. The analysis was performed by SPSS13.0 software.
Results
The growth of K562 cells was inhibited and the apoptosis of the cells was elevated by Curcumin in a dose-dependent and time-dependent manner, which was accompanied by the appearance of morphologic characteristics of apoptosis; After 24h of 100μM curcumin treatment,the percentage of apoptosis cells is (58.53±1.08)%; After 48h of 100μM curcumin treatment, the percentage of apoptosis cells is (84.70±1.53)%.,both of which increased significantly when compared with the untreated controls (P<0.05).Flow cytometry showed the cells were increase in G1 stage and decreased in S stage (P<0.05).Curcumin treatment decreased the mRNA and protein expression of BCL-XL,and increased mRNA and protein expression of BAK by Reverse transcription-polymerase chain reaction (RT-PCR).
Conclusion
1 Curcumin can induce apoptosis of K562 cells, there is a dose-dependent and time-dependent relationships.
2 The data showed Curcumin had the significant effect on K562 cell cycle, and hold back cell course from G1 stage to S stage,and induced K562 apoptosis.
3 Curcumin treatment decreased the mRNA and protein expression of BCL-XL,and increased mRNA and protein expression of BAK,which maybe is relevant to p53 signal transduction.
引文
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