青蒿琥酯诱导HL60细胞凋亡的研究.docVIP

青蒿琥酯诱导HL60细胞凋亡的研究 正文 青蒿琥酯诱导HL60细胞凋亡的研究 文1：青蒿琥酯诱导HL60细胞凋亡的研究 Apoptosis of HL60 Cells Induced by Artesunate In Vitro Abstract The study was aimed to investigate the effect of artesunate (ART) on the apoptosis of HL60 cells in vitro and the expression of Bcl2 and ICAD in the process of apoptosis induced by ART. The inhibition of ART on HL60 cells were evaluated by mea of MTT assay; cell apoptosis was detected by light microscopy, agarose gel electrophoresis, flow cytometry; Western blot was used to analyze the expression of Bcl2 and ICAD in cells during apoptosis induced by ART. The results showed that ART could significantly inhibit the proliferation of HL60 cells in timeand dosedependent manner. After treating HL60 cells with ART for 48 hou, the IC50 values was μg/ml and its inhibition effect contributed to the induced apoptosis. Bcl2 and ICAD protei both all expressed in HL60 cells, the level of expression declined as concentration increased. It is concluded that artesunate may induce apoptosis of HL60 cells in vitro, Bcl2 and ICAD may be an important control factor in the signal traduction pathway of ARTinduced apoptosis. Key words artesunate; HL60 cell line; apoptosis 青蒿琥酯（artesunate，ART），是具有倍半萜内酯类抗疟药青蒿素的衍生物，其除具有高效抗疟作用外，对多种肿瘤细胞均有诱导凋亡作用［1，2］。本研究观察了青蒿琥酯对HL60细胞的凋亡诱导作用及凋亡过程中bcl2和ICAD的表达情况，以便为青蒿琥酯作为抗白血病药物应用于临床提供理论基础。 材料和方法 药物和试剂 注射用青蒿琥酯为桂林南药股份有限公司产品，批号 041101，5%碳酸氢钠1 ml溶解后，用RPMI 1640配制所需浓度；碘化丙啶（PI）、噻唑蓝（MTT）为Sigma公司产品；细胞凋亡DNA Ladder提取试剂盒购自北京博大泰克生物基因技术有限公司；bcl2和ICAD单克隆抗体均为美国Santa Cruz公司产品。 细胞系及细胞培养 急性早幼粒细胞白血病细胞株HL60由浙江大学医学院附属第二 医院 肿瘤研究所提供。细胞按常规悬浮细胞培养法培养于含10%热灭活小牛血清的RPMI 1640培养液中，置37℃、5% CO2培养箱中培养。每2-3天传代1次，取对数生长期细胞进行实验。 MTT测定 取对数生长期细胞，调整细胞浓度至1×105/ml，接种于96孔板，每孔100 μl，青蒿琥酯终浓度分别为1、2、4、8、16、32、64及128 μg/ml，每孔100 μl，设空白对照组。药物分别作用24、48、72小时后，每孔加MTT 20 μl，于37℃、5% CO2培养箱中培养4小时，离心弃去上清液，每孔加入DMSO 150 μl，振荡摇匀，在570 nm酶标仪上测吸光度值（A570）， 计算 抑制率，求半数抑制浓度IC50。 细胞抑制率（%）=（A对照－A实验/A对照）×100% 细胞经对照组、给药组作用48小时后，收集细胞，经磷酸盐缓冲液（PBS，pH ）洗2遍后，细胞制片机离心、制片，WrightGiemsa染色，油镜下观察细胞形态