目的 探讨钩吻总碱对慢性粒细胞白血病K562细胞的抑杀作用及其机制，为其抗白血病研究提供科学依据。方法 以不同浓度钩吻总碱(25、50、100、200、400 μg·mL-1)干预K562细胞，MTT法测定其对K562细胞活力的影响;倒置相差显微镜观察其对K562细胞形态的影响;DAPI染色法观察K562细胞核形态变化;Annexin V FITC/PI流式细胞术检测K562细胞凋亡情况;比色法测定caspase-3活性变化;RT-PCR检测Bax和Bcl-2基因的表达水平。结果 钩吻总碱对K562细胞抑制效果呈时间、剂量依赖性，24 h时IC50为122 μg·mL-1;TAG干预K562细胞后细胞形态逐渐不规则、胞质不清、胞核皱缩，最终胞膜的完整性破坏;DAPI染色发现细胞体积变小，整体皱缩，胞核固缩、生成典型的凋亡小体;Annexin V FITC/PI流式细胞测定显示K562细胞凋亡以早凋为主，存在量效关系;不同浓度钩吻总碱作用于K562细胞24 h后caspase-3的活性提高;RT-PCR检测发现TAG干预后会下调Bcl-2基因表达、上调Bax基因表达，二者均表现出量效关系。结论 钩吻总碱可能通过上调Bax基因表达、下调Bcl-2基因表达，活化caspase-3，从而诱导慢性粒细胞白血病K562细胞发生早期凋亡，抑制其细胞活力。
Mechanism analysis of the apoptosis in chronic myeloid leukemia K562 cells induced by total alkaloids of Gelsemium elegans
Objective To investigate the inhibitory influence of total alkaloids of Gelsemium elegans(TAG)on chronic myeloid leukemia K562 cells and its mechanism,so as to provide experimental basis for the use of GE in the treatment of CML.Methods K562 cells were treated with different concentrations of TAG(25,50,100,200,400 μg·mL-1),and the effect of TAG on K562 cell viability was determined by MTT assay.The morphology of K562 cells was observed by inverted phase contrast microscope.DAPI staining was applied to observe the nuclear morphological changes of K562.Annexin V FITC/PI flow cytometry was used to detect the apoptosis of K562 cells.Caspase-3 activity was measured by colorimetry.RT-PCR was applied to detect the expression levels of Bax and Bcl-2 genes.Results The inhibitory effect of TAG on K562 cells was time and dose dependent,and the IC50 was 122 μg·mL-1 at 24 h;After the TAG intervened K562 cells,the cell morphology was gradually irregular,the cytoplasm was unclear,the nucleus was shrunk,and finally the integrity of the cell membrane was destroyed;DAPI staining showed that the cell volume was reduced and shrunk,the nucleus was pyknotic,and typical apoptotic bodies were formed;The results of Annexin V FITC/PI flow cytometry showed that the apoptosis of K562 cells was mainly early withering,and there was a dose effect relationship;The activity of caspase-3 in K562 cells was in-creased after 24 h treatment with disparate concentrations of TAG;RT-PCR testing report exhibit that expression of Bcl-2 was down regulated and Bax expression was up regulated,both of which showed a dose effect relationship.Conclusion The TAG can up regulate the expression of Bax gene,down regulate the expression of Bcl-2 gene,activate caspase-3,thereby inducing the early apoptosis of K562 cells and inhibiting their cell viability.
Total alkaloids of Gelsemium elegansChronic myeloid leukemiaK562 cellsApoptosis
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