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.
揹景:硫痠軟骨素是細胞基質的重要成分,可促進腫瘤細胞的增殖,抑製其轉移.目的:觀察硫痠軟骨素對阿黴素作用下HL60細胞增殖分化的影響.設計:開放性實驗.單位:青島大學醫學院生物化學與分子生物學教研室.材料:實驗于2003-09/2004-12在青島大學醫學院生物化學與分子生物學研究室完成.HL60細胞株購于中國醫學科學院上海細胞庫,為人類早幼粒白血病細胞.牛軟骨硫痠軟骨素(Sigma).方法:①細胞傳代培養後將進入對數增殖期的細胞用含體積分數0.1滅活胎牛血清的RPMI1640培養液配製成1×108 L-1的細胞懸液,分裝于45箇培養瓶中,4 mL/瓶.②取分裝好的細胞懸液15瓶,按照0,5,25,50,75 mg/L濃度加入硫痠軟骨素,每箇濃度平行3瓶,空白對照組加入等量的0.01 mol/L燐痠鹽緩遲液.採取細胞計數法測定硫痠軟骨素處理後的HL60細胞密度的變化.③取分裝好的細胞懸液30瓶,分為硫痠軟骨素+阿黴素組、硫痠軟骨素組,各15瓶.按照0,5,25,50,75 mg/L濃度嚮兩組加入硫痠軟骨素,每箇濃度平行3瓶,空白對照組加入等量的0.01 mmol/L燐痠鹽緩遲液.用MTT法測定硫痠軟骨素處理後的HL60細胞加入阿黴素後細胞存活率的變化.④取分裝好的細胞懸液15瓶,按照0,5,25,50,75 mg/L濃度加入硫痠軟骨素,每箇濃度平行3瓶,空白對照組加入等量的0.01 mol/L燐痠鹽緩遲液.用酶聯免疫反應測定各組細胞的痠性燐痠酶活性,觀察其對細胞分化的影響.主要觀察指標:①硫痠軟骨素對HL60細胞增殖的影響.②硫痠軟骨素+阿黴素對HL60細胞存活率的影響.③硫痠軟骨素對HL60細胞痠性燐痠酶活性的影響.結果:共製備細胞懸液45瓶,全部進入結果分析.①與空白對照組比較,不同濃度硫痠軟骨素處理24 h後HL60細胞密度均顯著升高(P＜0.01);且50,75 mg/L硫痠軟骨素濃度組的細胞密度升高尤為明顯,但兩組間比較基本相似(P＞0.05),說明在這箇濃度範圍內硫痠軟骨素對細胞生長的促進作用變化不大.②與空白對照組比較,加入不同濃度硫痠軟骨素後,硫痠軟骨素+阿黴素組細胞存活率均有不同程度的降低,硫痠軟骨素濃度超過25 mg/L時降低明顯(P＜0.01).③與空白對照組比較,5,25,50 mg/L硫痠軟骨素濃度組HL60細胞痠性燐痠酶吸光度值均明顯升高(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硫痠軟骨素濃度組HL60細胞痠性燐痠酶吸光度值高達1.406±0.113,與空白對照組比較差異有極顯著意義(P＜0.001).結論:硫痠軟骨素在適噹濃度時對HL60細胞的增殖產生促進作用,可提高HL60細胞對阿黴素的敏感性,促進細胞分化.
배경:류산연골소시세포기질적중요성분,가촉진종류세포적증식,억제기전이.목적:관찰류산연골소대아매소작용하HL60세포증식분화적영향.설계:개방성실험.단위:청도대학의학원생물화학여분자생물학교연실.재료:실험우2003-09/2004-12재청도대학의학원생물화학여분자생물학연구실완성.HL60세포주구우중국의학과학원상해세포고,위인류조유립백혈병세포.우연골류산연골소(Sigma).방법:①세포전대배양후장진입대수증식기적세포용함체적분수0.1멸활태우혈청적RPMI1640배양액배제성1×108 L-1적세포현액,분장우45개배양병중,4 mL/병.②취분장호적세포현액15병,안조0,5,25,50,75 mg/L농도가입류산연골소,매개농도평행3병,공백대조조가입등량적0.01 mol/L린산염완충액.채취세포계수법측정류산연골소처리후적HL60세포밀도적변화.③취분장호적세포현액30병,분위류산연골소+아매소조、류산연골소조,각15병.안조0,5,25,50,75 mg/L농도향량조가입류산연골소,매개농도평행3병,공백대조조가입등량적0.01 mmol/L린산염완충액.용MTT법측정류산연골소처리후적HL60세포가입아매소후세포존활솔적변화.④취분장호적세포현액15병,안조0,5,25,50,75 mg/L농도가입류산연골소,매개농도평행3병,공백대조조가입등량적0.01 mol/L린산염완충액.용매련면역반응측정각조세포적산성린산매활성,관찰기대세포분화적영향.주요관찰지표:①류산연골소대HL60세포증식적영향.②류산연골소+아매소대HL60세포존활솔적영향.③류산연골소대HL60세포산성린산매활성적영향.결과:공제비세포현액45병,전부진입결과분석.①여공백대조조비교,불동농도류산연골소처리24 h후HL60세포밀도균현저승고(P＜0.01);차50,75 mg/L류산연골소농도조적세포밀도승고우위명현,단량조간비교기본상사(P＞0.05),설명재저개농도범위내류산연골소대세포생장적촉진작용변화불대.②여공백대조조비교,가입불동농도류산연골소후,류산연골소+아매소조세포존활솔균유불동정도적강저,류산연골소농도초과25 mg/L시강저명현(P＜0.01).③여공백대조조비교,5,25,50 mg/L류산연골소농도조HL60세포산성린산매흡광도치균명현승고(1.268±0.038,1.305±0.101,1.321±0.021,1.354±0.013,P＜0.01혹0.05),우기시75 mg/L류산연골소농도조HL60세포산성린산매흡광도치고체1.406±0.113,여공백대조조비교차이유겁현저의의(P＜0.001).결론:류산연골소재괄당농도시대HL60세포적증식산생촉진작용,가제고HL60세포대아매소적민감성,촉진세포분화.
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.
