苦参碱诱导胶质瘤C6细胞凋亡及其可能的基因机制

Matrine induces apoptosis of glioma cell C6 and the possible mechanism

目的:探讨苦参碱诱导C6胶质瘤细胞凋亡的作用特点和可能的基因作用机制。方法:MTT法测定不同浓度苦参碱对C6细胞增殖的抑制率,求出IC50值;倒置显微镜及透射电镜观察苦参碱诱导C6细胞凋亡的形态学改变;Annexin/FITC染色流式细胞术检测不同浓度苦参碱诱导C6细胞的凋亡率;实时定量PCR芯片(Real-time PCR Array)检测苦参碱作用后C6细胞凋亡相关基因的差异表达;免疫细胞化学及Western blotting方法检测苦参碱对C6细胞caspase-3表达的影响。结果:苦参碱对C6细胞增殖的抑制率随着药物剂量的增加(0.1～1.0mg/ml)而增加(P<0.05),其IC50为0.715mg/ml。倒置显微镜观察显示苦参碱可诱导胶质瘤细胞出现凋亡改变,透射电镜观察显示苦参碱诱导胶质瘤细胞出现凋亡的超微形态改变,流式细胞术检测显示胶质瘤细胞的凋亡率随着药物剂量的增加(0.2～1.0mg/ml)而增大[(3.56±0.73)%～(27.55±1.92)%](P<0.05)。胶质瘤细胞经苦参碱作用后出现显著表达差异基因共68个,其中57个基因表达明显上调、11个基因表达明显下调,其中有22个基因与诱导凋亡的死亡受体相关,15个基因与线粒体途径有关;ICC及Western blotting检测都显示苦参碱可诱导C6胶质瘤细胞caspase-3表达的上调(P<0.05)。结论:苦参碱能诱导C6胶质瘤细胞的凋亡,其机制与死亡受体途径和线粒体途径的众多基因有关。

Objective：To explore the apoptosis inducing effect of matrine on C6 glioma cells and the related mechanisms. Methods： MTT assay was used to examine the inhibition of C6 glioma cell line by matrine at various concentrations and the IC50 was calculated. Inverted microscope and TEM （transmission electron microscope） were employed to observe the morphological alterations of C6 glioma cells after exposure to matrine; FCM （Flow cytometry） was used to detect the apoptosis rate of C6 glioma cells; and real-time PCR was used to examine the differential expression of related genes. ICC and Western blotting was used to detect the expression of caspase-3. Results ： MTT showed that the cell inhibition effect of matrinc increased with its concentration （0.1-1.0 mg/ml） （ P 〈 0.05 ）, with the IC50 being 0.715 mg/ml. Inverted microscope and TEM showed that matrine induced apoptosis of C6 glioma cells. FCM showed that the apoptosis rate increased [ （3- 56 ± 0.73 ） % - （27.55 ± 1.92 ） % ] with the increase of matrine concentration （0.2 - 1.0 mg/ml） （ P 〈 0.05 ）. Real-time PCR showed that matrine induced up-regulation of 58 genes and down-regulation of 11 genes, and 22 of them were related to apoptosis-inducing death receptor, 15 of them to apoptosis-inducing mitochondrial pathway. ICC and Western blotting showed that matrine induced up-regulation of caspase-3 expression （ P 〈 0.05 ）. Conclusion： Matrine can induce C6 glioma cell apoptosis and the mechanism might be related to a number of genes involved in death receptor pathway and mitochondrial pathway.