5-氟尿嘧啶损伤骨髓基质细胞致造血细胞应激诱导性早衰  

5-FU-Injured Bone Marrow Stromal Cells Initiate Stress-induced Premature Senescence of Hematopoietic Cells

作　　者：熊丽溶 宋小英[1] 景鹏伟 王亚平[1] 王璐[1] 

机构地区：[1]重庆医科大学干细胞与组织工程学研究室组织学胚胎学教研室,重庆400016

出　　处：《中国实验血液学杂志》2017年第4期1178-1186,共9页Journal of Experimental Hematology

基　　金：国家自然科学基金资助项目(81173398);重庆市科委基础与前沿研究资助项目(cstc2014jcyj A10001)

摘　　要：目的:探讨抑瘤浓度下的5-氟尿嘧啶(5-fluorouracil,5-FU)对人骨髓基质细胞是否有损伤作用以及该作用对造血细胞的影响。方法:应用CCK-8法测定乳腺癌细胞株MCF-7、结肠癌细胞株HCT-116及人骨髓基质细胞株HS-5对不同浓度5-FU的敏感性。5-FU作用HS-5后,结晶紫染色计数成纤维细胞集落;流式细胞术分析细胞周期;Annexin V/PI双染及Hoechest染色检测细胞凋亡;DCFH-DA法检测胞内活性氧(reactive oxygen species,ROS)水平;ELISA及免疫荧光法检测细胞因子KL、GM-CSF、RANTS、SDF水平。人脐血单个核细胞(human umbilical cord blood mononuclear cell,h UCB-M NC)与HS-5共培养后,台盼蓝染色计数h UCB-M NC;流式细胞术检测细胞周期、ROS水平、CD34+细胞百分率;酶学法检测谷胱甘肽过氧化物酶(Glutathione peroxidase,GSH-Px)和超氧化物歧化酶(Superoxide dismutase,SOD)含量;β-半乳糖苷酶染色检测衰老的h UCB-MNC。结果:5-FU 12.5-100μg/ml对M CF-7、HCT-116和HS-5均有增殖抑制作用,且该作用具有浓度依赖性和时间依赖性,其中HS-5对5-FU更为敏感。5-FU作用后HS-5细胞周期阻滞,凋亡率上升,胞内ROS含量显著升高,造血生长因子分泌降低,炎性趋化因子升高。与经5-FU作用的HS-5共培养后,h UCB-MNC数量及CD34+细胞比例降低,G1期阻滞,细胞抗氧化能力降低,胞内ROS含量显著上升,衰老的造血细胞增多。结论:5-FU可导致骨髓基质细胞氧化损伤、分泌生物活性物质改变,诱发造血细胞氧化应激性早衰。Objective： To investigate the damage effect of 5-fluorouracil （5 - FU） with tumor inhibition concentration on human bone marrow mesenchymal stem cells （hBMSC） and influence of its effect on the hematopoietic cells. Methods： The Cell Counting Kit-8 was used for determining the sensitivity of breast cancer cell line MCF-7, colon cancer cell line HCT-116 and HS-5 derived from human bone marrow stronal cell line to the different doses of 5- fluorouracil in vitro. After HS-5 was treated with 5-fluorouracil, crystal violet staining assay was used to count the number of colony forming unit-fibroblast, the distribution of cell cycle was analyzed by flow cytometry （FCM）, apoptosis was assessed by Annexin V/PI double-stained method and Hoechest staining; DCFH-DA staining was used to analyse the level of reactive oxygen species （ ROS）, ELISA and immuofluorescence were used to detect cytokines KL, GM-CSF, RANTS and SDF. The hUCB-MNC was counted by trypan blue staining after co-culture with HS-5, FCM was used to detect the cell cycle distribution, ROS level and the ratio of CD34 ~ cells. The levels of glutathione peroxidase （GSH-Px） and total superoxide dismutase （T-SOD） were measured by enzymatic assay. The senescence associated-13-galactosidase （SA-13-Gal） staining was used to detect the senescent hUCB-MNC. Results： 5-Fluorouracil of 12. 5 μg/ml - 100 μg/ml inhibited the proliferation of MCF-7, HCT-116 and HS-5 cells in dose-dependent and time- dependent manner, among them HS-5 was more sensitive to 5- fluorouracil. After treatment with 5-fluorouracil, the HS- 5 cell cycle was blocked. The apoptosis rate and the intracellular ROS level of HS-5 significantly increased. Also HS-5- secreted hematopoietic growth factors decreased and inflammatory chemokines increased. After co-cultured with 5- fluorouracil-treated HS-5, the number of hUCB-MNC and the ratio of CD34＋ cells were decreased, hUCB-MNC cell cycle blocked in G1 phase. The antioxidant capacity also decreased and the intracellular RO

关 键 词：5-氟尿嘧啶 骨髓基质细胞 脐血单个核细胞 氧化应激 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]