青蒿琥酯对原代慢性粒细胞白血病细胞生长抑制、凋亡诱导及bcr/abl融合基因、P210蛋白、磷酸化SHP-2蛋白表达的影响

Effects of Artesunate on Growth Inhibition,Apoptosis Induction and Expression of bcr/abl Fusion Gene,P210 and Phosphorylation of SHP-2 Protein in Primary Chronic Myeloid Leukemia Cells

目的探讨青蒿琥酯对原代慢性粒细胞白血病细胞生长抑制、凋亡诱导及bcr/abl融合基因、P210蛋白、Src同源区2含域磷酸酶-2(SHP-2)蛋白磷酸化的影响。方法分离培养原代慢性粒细胞白血病细胞,采用不同浓度青蒿琥酯(0、5、10、20μg·mL^(-1))进行干预,四甲基偶氮唑盐比色法(MTT)检测细胞生长抑制情况;细胞计数法检测细胞生存情况;罗丹明123染色法检测细胞线粒体膜电位的变化;电镜观察细胞超微结构的变化;流式细胞术(FCM)检测青蒿琥酯对细胞凋亡及胀亡的影响;荧光定量聚合酶链式反应(RT-PCR)检测bcr/abl融合基因mRNA表达;Western Blot检测P210、SHP-2及磷酸化SHP-2蛋白表达。结果青蒿琥酯可抑制原代慢性粒细胞白血病细胞的生长,降低细胞存活率和线粒体膜电位水平(P<0.05),且与药物浓度和时间呈现一定的依赖性;不同浓度药物和不同作用时间可导致细胞凋亡率及胀亡率明显升高(P<0.01),同时可降低bcr/abl融合基因mRNA、P210、SHP-2和磷酸化SHP-2蛋白的表达水平(P<0.05,P<0.01)。结论青蒿琥酯可通过抑制原代慢性粒细胞白血病细胞的生长,促进其凋亡和胀亡,以及降低bcr/abl融合基因mRNA、P210、SHP-2、磷酸化SHP-2蛋白在原代慢性粒细胞白血病细胞中的表达水平,达到治疗慢性粒细胞白血病(CML)的目的。

Objective To investigate the effects of artesunate on cell growth inhibition,apoptosis induction and bcr/abl fusion gene,P210 and phosphorylation of Src homologous region 2 domain Phosphatase-2(SHP-2)protein in primary chronic myeloid leukemia(CML).Methods The primary chronic myelogenous leukemia cells were isolated and cultured.Artesunate of different concentrations(0,5,10,20μg·mL-1)was applied to the cells.Growth inhibition was detected by methylthiazole-tetrazole(MTT)colorimetry assay;cell survival was measured by cell counting;mitochondrial membrane potential was measured by Rhodamine 123 staining;ultrastructure was observed by electron microscopy.The flow cytometry(FCM)was used to detect the apoptosis and oncosis of cells.Fluorescent quantitative polymerase chain reaction(RT-PCR)was used to detect the expression of bcr/abl fusion gene.Western Blot was used to detect the expression of P210,SHP-2 and phosphorylated SHP-2 protein.Results Artesunate can inhibit the growth of primary chronic myeloid leukemia cells,reduce the cell survival rate and mitochondrial membrane potential level(P<0.05),which is dependent on the concentration and time exposure of drugs.Different concentrations of drugs and different time of action can lead to a significant increase in apoptotic rate and oncosis rate(P<0.01),and can reduce expression levels of bcr/abl fusion gene mRNA,P210,SHP-2 and phosphorylated SHP-2 protein at the same time(P<0.05 or P<0.01).Conclusion Artesunate can treat CML by inhibiting the growth of primary chronic myeloid leukemia cells,promoting apoptosis and oncosis,and reducing the expression levels of bcr/abl fusion gene,P210,SHP-2 and phosphorylated SHP-2 protein in chronic myeloid leukemia(CML)cells.