痰热清注射液对急性T淋巴细胞白血病Molt4细胞系生物学活性的抑制作用及其机制

本研究探讨痰热清注射液体外对人急性T淋巴细胞白血病Molt4细胞系生物学活性的影响及其作用机制。将痰热清注射液倍比稀释成1:2、1:4、1:8、1:16、1:32、1:64、1:128、1:256、1:512,共9个浓度组,处理增殖期Molt4细胞,镜下观察不同时间点各浓度组细胞生长情况;应用CCK-8法检测细胞增殖能力,绘制生长曲线,计算抑制率和半数抑制浓度(IC50);分别采用PI染色法和PI/Annexin V双染法,通过流式细胞术检测Molt4细胞周期及凋亡的变化;采用实时定量PCR方法分析痰热清注射液处理Molt4细胞后凋亡相关基因caspase-3和bcl-2的表达量变化。结果表明:痰热清注射液对Molt4细胞增殖具有抑制作用,1:2至1:16稀释浓度的细胞毒性大,细胞基本死亡;抑制作用呈剂量依赖性,IC50为1:142稀释浓度。痰热清注射液1:32浓度处理72小时Molt4细胞处于S期的数量明显减少(p<0.05),凋亡细胞比例明显增加(p<0.05);同时,caspase3表达升高和bcl-2表达明显减低(p<0.05)。结论:痰热清注射液可抑制Molt4细胞系增殖、促进其凋亡,其机制可能是通过减少S期细胞,下调bcl-2基因和上调caspase3基因而实现。

Inhibitory Effect of Tanreqing Injection on Proliferation of T Lymphoblastic Leukemia Cell Line Molt4 In Vitro and Its Mechanism

This study was purposed to explore the effects of Tanreqing injection on the biologic activities of human acute T lymphoblastic leukemia cell line Molt4 in vitro and its mechanism.Tanreqing injection was proportionally diluted and divided into 9 groups of different concentrations,including 1∶2,1∶4,1∶8,1∶16,1∶32,1∶64,1∶128,1∶256 and 1∶512.Molt4 cells were treated with those different concentrations of Tanreqing injection,and the cell growth status at various time points of different concentrations was observed under microscope.CCK-8 assay was employed to detect ability of cell proliferation,growth curve was drawn,the inhibition ratio and 50% inhibiting concentration（IC50） were calculated.Flow cytometry with PI and PI/Annexin V double stainings were used to detect the cell cycle and apoptosis of Molt4 cells after the treatment of Tanreqing injection respectively.Caspase-3 and Bcl-2 mRNA expression of Molt4 cells were determined by real-time quantitative PCR.The results showed that Tanreqing injection displayed an inhibitory effect on the proliferation of Molt4 cell line.In 1∶2,1∶4,1∶8 and 1∶16 concentration groups,great cytotoxicity was observed and numerous cells were dead.The inhibitory effect of Tanreqing injection was dose-dependent.IC50 was 1∶142 dilution concentration.In the 1∶32 concentration group,S phase cell quantity remarkably decreased（p0.05） and apoptosis rate significantly increased（p0.05） at 72 hours after Tanreqing injection treatment.Simultaneously,caspase-3 mRNA expression increased and Bcl-2 mRNA expressions was downregulated（p0.05）.It is concluded that the Tanreqing injection has an inhibitory effect on Molt4 cell proliferation and promotes its apoptosis.These biological effects of Tanreqing injection are partly related to cell reduction in S phase,downregulation of bcl-2 gene and upregulation of caspase 3.