穿心莲内酯对急性淋巴细胞白血病细胞的生长抑制及凋亡诱导作用  

Growth-inhibitory and apoptosis-inducing effects of andrographolide on acute lymphoblastic leukemia cells

作　　者：李晓文 周越菡[1] 邓健志[2] 罗珍华 江海键 张漫丽 覃楚玲 LI Xiao-wen;ZHOU Yue-han;DENG Jian-zhi;LUO Zhen-hua;JIANG Hai-jian;ZHANG Man-li;QIN Chu-ling(Guilin Medical University,Guilin 541100,China;Guilin University of Technology,Guilin 541004,China)

机构地区：[1]桂林医学院,广西桂林541100 [2]桂林理工大学,广西桂林541004

出　　处：《中国病理生理杂志》2020年第6期977-984,共8页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81660031,No.81360090);广西自然科学基金资助项目(No.2014GXNSFBA118151);广西自治区级大学生创新创业项目(No.201810601084)。

摘　　要：目的:研究穿心莲内酯(andrographolide,AND)对急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)细胞生长和凋亡的影响。方法:人ALL细胞株CEM-C1经AND处理12 h、24 h和48 h,应用CCK-8法检测AND对细胞活力的影响;应用Wright-Giemsa染色法观察AND处理24 h对CEM-C1细胞形态及数量的影响;应用流式细胞术+annexin V-FITC/碘化丙啶(propidium iodide,PI)双染色法分析AND作用24 h对CEM-C1细胞凋亡的作用;采用流式细胞术+PI染色法分析AND作用24 h对CEM-C1细胞周期分布的作用;用Western blot法检测AND对CEM-C1细胞凋亡相关蛋白水平的影响;应用JC-1线粒体膜电位(mitochondrial membrane potential,MMP)检测试剂盒检测AND作用24 h对CEM-C1细胞MMP的影响。最后将CEM-C1细胞接种于BALB/c-nu雌性裸鼠右胁腹侧建立裸鼠ALL移植瘤模型,通过检测肿瘤体积及重量观察AND的体内抗ALL作用,并应用免疫组化法检测裸鼠肿瘤组织凋亡蛋白cleaved caspase-3的蛋白水平。结果:CCK-8实验结果表明,AND对CEM-C1细胞活力的抑制作用呈时间及剂量依赖性。Wright-Giemsa染色结果显示,应用AND后CEM-C1细胞缩小、凝聚,细胞染色加深,细胞质呈均质红染并且细胞数减少。流式细胞术检测结果表明,AND处理细胞24 h后,随着AND浓度的增加,凋亡细胞数量增多,且细胞周期被阻滞在G2期,细胞生长被抑制。Western blot结果显示,AND上调活化凋亡蛋白cleaved caspase-3、cleaved caspase-7及促凋亡蛋白Bax的水平,并下调抗凋亡蛋白Bcl-2的表达;且cleaved caspase-3/caspase-3、cleaved caspase-7/caspase-7和Bax/Bcl-2的比值随AND浓度升高而增大。JC-1染色法结果表明,CEM-C1细胞的MMP随AND剂量增大而降低。体内实验结果显示,AND明显抑制荷瘤鼠的肿瘤生长,并上调肿瘤组织cleaved caspase-3的水平。结论:穿心莲内酯在体内外均抑制ALL细胞的生长并诱导其发生凋亡。AIM:To explore the effect of andrographolide(AND)on the growth and apoptosis of acute lym⁃phoblastic leukemia(ALL)cells.METHODS:CCK-8 assay was used to assess the viability of human acute lymphoblas⁃tic leukemia CEM-C1 cells treated with AND for 12 h,24 h and 48 h.The cell morphological changes were observed by Wright-Giemsa staining.The cell apoptosis was detected by flow cytometry with annexin V-FITC/propidium iodide(PI)staining,and the cell cycle was examined by flow cytometry with PI staining.The expression levels of apoptosis-related proteins were examined by Western blot.The mitochondrial membrane potential(MMP)of the cells was determined by JC-1 assay.RESULTS:The results of CCK-8 assay indicated that AND inhibited the viability of CEM-C1 cells in a dose and time-dependent manner.After administration of AND for 24 h,CEM-C1 cells shrank,the cytoplasm turned red and the cell numbers were significantly reduced.Incubation of AND for 24 h resulted in G2-phase arrest and apoptosis.Treat⁃ment with AND for 24 h increased the protein levels of cleaved caspase-3,cleaved caspase-7 and Bax,and down-regulated Bcl-2 in the CEM-C1 cells(P<0.05).The ratios of cleaved caspase-3/caspase-3,cleaved caspase-7/caspase-7 and Bax/Bcl-2 were elevated with the increase in the concentration of AND.Collapsed MMP in CEM-C1 cells was observed after AND administration for 24 h.Treatment with AND in vivo suppressed the growth of the xenograft tumor and increased the protein level of cleaved caspase-3.CONCLUSION:Andrographolide exerts growth-inhibitory and apoptosis-inducing ef⁃fects on ALL cells both in vitro and in vivo.

关 键 词：急性淋巴细胞白血病 穿心莲内酯 细胞生长 细胞凋亡 线粒体膜电位 

分 类 号：R733.7[医药卫生—肿瘤] R363.2[医药卫生—临床医学]