【作者】 夏靖燕；

【导师】 赵小英；

【作者基本信息】 浙江大学 ， 内科学， 2003， 硕士

【摘要】 背景 紫杉醇(Paclitaxel，taxol)是一种作用于微管的广谱抗癌药，目前已在进展期恶性实体瘤如卵巢癌、乳腺癌和肺癌中得到了广泛运用，但国内尚未见其应用于临床治疗白血病的报道，已有体外实验证实紫杉醇对包括K562、HL-60等多种白血病细胞具有增殖抑制作用，可用于治疗急性白血病，但国外Ⅰ期临床研究的报道其单独治疗白血病的结果仍不令人满意，目前关于紫杉醇与常用白血病化疗药物如阿霉素、阿糖胞苷联合应用对白血病细胞的增殖抑制作用未见报道。本实验旨在研究紫杉醇和阿霉素、阿糖胞苷单独和联合应用对K562、HL-60细胞的增殖抑制作用，以便为临床用药提供依据。 材料与方法 紫杉醇注射液由四川太极制药有限公司惠赠；阿霉素为浙江海正药业股份有限公司产品；阿糖胞苷为上海华联制药有限公司产品。人急性红白血病细胞株K562细胞、急性粒细胞白血病细胞株HL-60细胞均由浙江大学医学院肿瘤研究所提供。本实验分单一用药实验和联合 浙江大学硕士学位论文用药实验两部分，单一药物作用实验设紫杉醇实验组、阿霉素实验组、阿糖胞昔实验组和空白对照组，紫杉醇的浓度范围为 0．125＊ u g／mL、阿霉素的浓度范围为 0．31 25J u g／mL、阿糖胞苦的浓度范围为 264u g／mL。联合药物作用实验分为 4组即紫杉醇邓 霉素组、紫杉醇叩糖胞苦组、紫杉醇＋阿霉素＋阿糖胞昔组和空白对照组。根据单一用药对 K562、HL60作用的实验结果，选择抑制率在 10刁0o左右的阿霉素、阿糖胞昔浓度作为联合用药的浓度，阿霉素浓度为 0．3 u g／mL，阿糖胞昔浓度为 2 u g／mL，在各组加入不同浓度的紫杉醇。药物作用时间均为72h，分别在作用24、48、72h后用MTT法测定紫杉醇、阿霉素、阿糖胞昔单独和联合应用对K562、HL．60细胞的增殖抑制作用，计算抑制率并求得半数抑制浓度门C50入实验结果用SPSS10刀for windows统计软件分析处理，IC50采用加权直线线性回归法求得，两组间抑制率比较采用矿检验，以P＜o．05为差异具有显著性意义。 结 果 单独用药实验结果示，紫杉醇、阿霉素和阿糖胞苦对K562、HL60均有不同程度的增殖抑制作用，并呈明显的浓度．效应和时间－效应关系。紫杉醇、阿霉素和阿糖胞昔作用 K562细胞 72h的 IC50分别为 0．11n g／mL、0．18 u g／mL、3．71u g／mL，作用 HL－60细胞 72h的 IC50分别为 0．37 u g／mL、0．22 u g／mL、1．93 u g／mL。由此可见紫杉醇对 K562细胞的抑制作用大于HL60细胞。 联合用药实验结果显示，紫杉醇联用阿霉素或阿糖胞昔或三药联用后对K562、HL60 细胞株的抑制率显著增加，明显高于单一用药（P＜0刀5人 三药联用组的抑制作用也大于两药联用组，与紫杉醇联用阿糖胞昔组相比其抑制率有显著差异什＜0刀5X但与紫杉醇联用阿霉素组相比无显著差异仔＞0刀5X提示三药联用组作用与紫杉醇联用阿霉素组相近。当 0．3 n g／mL阿霉素联用 0．125 u g／mL紫杉醇后其对 K562的抑制率能从25．31％提高到55．49％，其作用效果相当于单用阿霉素两倍 二 浙江大学硕士学位论文剂量的作用强度，当紫杉醇浓度提高到 0．5 n g／mL，其作用相当于单用阿霉素八倍剂量的作用强度。 结 论 紫杉醇、阿霉素和阿糖胞昔对K562、HL－60均有一定的增殖抑制作用，并呈时间和浓度依赖性。紫杉醇对K562 细胞的抑制作用大于HL60细胞，紫杉醇与阿霉素或阿糖胞苦联合应用可明显增强对K562、HL＊0 的增殖抑制作用，小剂量的紫杉醇即可明显提高阿霉素的作用强度。我们认为紫杉醇可作为白血病治疗中可选择的药物之一，联合运用紫杉醇与阿霉素可望成为治疗白血病新的有效的方案。更多还原

【Abstract】 Paclitaxel(Taxol) is one kind of broad-spectrum anti-tumor drugs, which has an effect on microtubule. It has been shown to be widely used in the treatment of patients with advanced malignant solid tumors such as ovary cancer, breast cancer and lung cancer, but there has been no report about its application in clinical treatment of leukemia in china. Although it was reported that taxol could inhibit the proliferation of many leukemia cell lines including K562, HL-60 and so on in vitro, the result of a phase I clinical study on the application of taxol alone at abroad was unsatisfactory. No report has covered the effects of the combination of taxol with some common chemotherapeutic drugs, such as adriamycin (ADM), cytosine arabinoside (Ara-C) on proliferation of human leukemia cells. The present study aims to observe the anti-proliferation effects of the combination of taxol with ADM, Ara-C and each agent alone on leukemia cell lines K562 and HL-60 in vitro, which may providesome clues to clinical application.Materials and MethodsThe human acute erythroleukemia cell line K562 and human acute granulocytic leukemia cell line HL-60 were provided by the Cancer Institute of College of Medicine of ZheJiang University. Taxol was provided by Sichuan Taiji Pharmaceutical Co Ltd, ADM was provided by Zhejiang Haizheng Pharmaceutical Co Ltd, Ara-C was provided by Shanghai Hualian Pharmaceutical Co Ltd. This study was divided into two parts. One was single agent experiment, and the other was agents combination experiment. There were four groups for single agent experiment: taxol group with the concentration of 0.125-4μ g/mL, ADM group with the concentration of 0.3125-10 μ g/mL, Ara-C group with the concentration of 2-64 μ g/mL and negative control group. There were also four groups for agents combination: taxol+ADM group, taxol+Ara-C group, taxol+ADM+Ara-C group and negative control group. According to the results from single agent experiment, we chose 0.3 μ g/mL ADM and 2 μ g/mL Ara-C in which concentration their inhibition rates on K562 or HL-60 were about 10-30%, and different concentrations of taxol. After the cells had been cultured for 24, 48 and 72 hours, we detected the anti-proliferation effects of the agents on the cell lines in all groups by method of MTT, then calculated the inhibition rate, figured out the 50% inhibition concentration (IC50) value. The statistic precession was carried out in SPSS 10.0 for windows. IC50 value was figured out by linear regression. Comparison with inhibition rates between two groups was performed by Mest and the P value less than 0.05 was considered statistically significant.ResultsSingle agent experiment showed that taxol, ADM and Ara-C had various degrees of inhibitory effects on the cell lines K562, HL-60 and their inhibition rates had obvious concentration-effect and time-effect relationship. The IC50 values of taxol, ADM and Ara-C on K562 after 72h were 0.11 μ g/mL, 0.18 μ g/mL and 3.71 μ g/mL respectively, and those on HL-60 were 0.37 μ g/mL, 0.22 μ g/mL and 1.93 μ g/mL respectively. From the above data, the inhibitory effect of taxol on K562 is much stronger than that on HL-60.Agents combination experiment showed that compared with taxol alone, combination of taxol with ADM or Ara-C or both of them has a remarkable increase in anti-proliferation activity on cell lines K562 and HL-60 (P<0.05) . Although the highest inhibitory effect was observed in the combination of three agents, but there was no significant difference between taxol with ADM and three agents (P>0.05), and the difference between taxol with Ara-C and three agents was significant (P<0.05). It suggested that the effect of the combination of three agents was similar to that of taxol with ADM. When 0.3 μ g/mL ADM was combined with 0.125 μ g/mL taxol, the inhibition rate rose from 25.31% to 55.49% and this effect was equal to that of double-concentration of ADM alone. When the concentration of taxol rose to 0.5 u g/mL, its inhibitory effect with ADM was equal to that of eigh更多还原