硫酸软骨素对HL60细胞增殖分化的影响

摘要

背景:硫酸软骨素是细胞基质的重要成分,可促进肿瘤细胞的增殖,抑制其转移.目的:观察硫酸软骨素对阿霉素作用下HL60细胞增殖分化的影响.设计:开放性实验.单位:青岛大学医学院生物化学与分子生物学教研室.材料:实验于2003-09/2004-12在青岛大学医学院生物化学与分子生物学研究室完成.HL60细胞株购于中国医学科学院上海细胞库,为人类早幼粒白血病细胞.牛软骨硫酸软骨素(Sigma).方法:①细胞传代培养后将进入对数增殖期的细胞用含体积分数0.1灭活胎牛血清的RPMI1640培养液配制成1×108 L-1的细胞悬液,分装于45个培养瓶中,4 mL/瓶.②取分装好的细胞悬液15瓶,按照0,5,25,50,75 mg/L浓度加入硫酸软骨素,每个浓度平行3瓶,空白对照组加入等量的0.01 mol/L磷酸盐缓冲液.采取细胞计数法测定硫酸软骨素处理后的HL60细胞密度的变化.③取分装好的细胞悬液30瓶,分为硫酸软骨素+阿霉素组、硫酸软骨素组,各15瓶.按照0,5,25,50,75 mg/L浓度向两组加入硫酸软骨素,每个浓度平行3瓶,空白对照组加入等量的0.01 mmol/L磷酸盐缓冲液.用MTT法测定硫酸软骨素处理后的HL60细胞加入阿霉素后细胞存活率的变化.④取分装好的细胞悬液15瓶,按照0,5,25,50,75 mg/L浓度加入硫酸软骨素,每个浓度平行3瓶,空白对照组加入等量的0.01 mol/L磷酸盐缓冲液.用酶联免疫反应测定各组细胞的酸性磷酸酶活性,观察其对细胞分化的影响.主要观察指标:①硫酸软骨素对HL60细胞增殖的影响.②硫酸软骨素+阿霉素对HL60细胞存活率的影响.③硫酸软骨素对HL60细胞酸性磷酸酶活性的影响.结果:共制备细胞悬液45瓶,全部进入结果分析.①与空白对照组比较,不同浓度硫酸软骨素处理24 h后HL60细胞密度均显著升高(P＜0.01);且50,75 mg/L硫酸软骨素浓度组的细胞密度升高尤为明显,但两组间比较基本相似(P＞0.05),说明在这个浓度范围内硫酸软骨素对细胞生长的促进作用变化不大.②与空白对照组比较,加入不同浓度硫酸软骨素后,硫酸软骨素+阿霉素组细胞存活率均有不同程度的降低,硫酸软骨素浓度超过25 mg/L时降低明显(P＜0.01).③与空白对照组比较,5,25,50 mg/L硫酸软骨素浓度组HL60细胞酸性磷酸酶吸光度值均明显升高(1.268±0.038,1.305±0.101,1.321±0.021,1.354±0.013,P＜0.01或0.05),尤其是75 mg/L硫酸软骨素浓度组HL60细胞酸性磷酸酶吸光度值高达1.406±0.113,与空白对照组比较差异有极显著意义(P＜0.001).结论:硫酸软骨素在适当浓度时对HL60细胞的增殖产生促进作用,可提高HL60细胞对阿霉素的敏感性,促进细胞分化.%BACKGROUND: Chondroitin sulfate is the important component of cell matrix, it can accelerate the proliferation of tumor cells and restrain its ransfer.OBJECTIVE: To observe the effects of chondroitin sulfate on the proliferation and differentiation of HL60 cells under the action of adriamycin.DESIGN: An open experiment.SETTING: Department of Biochemistry and Molecular Biology, Medical College of Qingdao University.MATERIALS: The experiments were carried out in the Research Room of Biochemistry and Molecular Biology, Medical College of Qingdao University of September 2003 to December 2004. Experimental materials and reagents: HL60 cell strains, which were the cells from promylocytic leukemia, were purchased from Shanghai Cell Bank, Chinese Academy of Medical Sciences; Bovine cartilage chondroitin sulfate (Sigma) was also used.METHODS: ① After the passage and culture, the cells at the logarithmic proliferative phase were dispensed into cell suspension of 1×108 L-1 with RPMI1640 culture medium containing inactivated fetal bovine serum of 0.1in volume fraction, and then filled into the culture bottles with 4 mL in each bottle for a total of 45 bottles. ② Chondroitin sulfate was added to 15 bottles filled with cell suspension according to the concentrations of 0, 5,25, 50 and 75 mg/L respectively, and 3 bottles for each concentration, and 0.01 mol/L phosphate buffer (pH7.2) was added in the blank control group. Then the density of HL60 cells was determined by cell counting after treatment of chondroitin sulfate. ③ Thirty bottles filled with cell suspension were divided into chondroitin sulfate+adriamycin group and chondroitin sulfate group, 15 bottles in each group. Chondroitin sulfate of 0, 5,25, 50 and 75 mg/L was added to the two groups, and 3 bottles for each concentration, and 0.01 mol/L phosphate buffer (pH7.2) wass adokd in the blank control group. Then the survival rate of chondroitin sulfate treated HL60 was detected after adding adriamycin. ④ Chondroitin sulfate was added to 15 bottles filled with cell suspension according to the concentrations of 0, 5, 25, 50 and 75 mg/L respectively, and 3 bottles for each concentration, 0.01 mol/L phosphate buffer (pH7.2) was added in the blank control group. The activity of acid phosphatase was detected with enzymelinked immunosorbant assay (ELISA) in each group, and the effect of the cell differentiation was observed.MAIN OUTCOME MEASURES: ① Effects of chondroitin sulfate on the proliferation of HL60 cells; ② Effects of chondroitin sulfate plus adriamycin on the survival rate of HL60 cells; ③ Effect of chondroitin sulfate on the activity of acid phosphatase of HL60 cells.RESULTS: Totally 45 bottles of cell suspension were prepared, and all were involved in the analysis of results. ① As compared with the blank control group, the densities of HL60 cells at 24 hours after treated with chondroitin sulfate of different concentrations were all significantly increased (P ＜ 0.01), which were increased more obviously in the 50 and 75 mg/L chondroitin sulfate treated groups, and there was no significant difference between the two groups (P ＞ 0.05), which indicated that chondroitin sulfate within the range of concentration did not accelerate the growth of cells greatly. ② As compared with the blank control group, the survival rates of HL60 cells in the chondroitin sulfate+adriamycin groups were decreased to different extents after chondroitin sulfate of different concentrations were added, and it decreased obviously when the concentration of chondroitin sulfate was higher than 25 mg/L (P ＜ 0.01). ③ As compared with the blank control group, the A values of acid phosphatase of the HHL60 cells were all obviously increased in the 5, 25 and 50 mL chondroitin sulfate treated groups (1.268±0.038, 1.305±0.101, 1.321±0.021,1.354±0.013, P ＜ 0.01 or 0.05), especially that it reached 1.406±0.113 in the 75 mL chondroitin sulfate treated group, which was extremely and significantly different from that in the blank control group (P ＜ 0.001).CONCLUSION: Chondroitin sulfate with a proper concentration can accelerate the proliferation of HL60 cells, and it can increase the sensibility of HL60 cells to adriamycin, and promote the differentiation of HL-60 cells.