骨髓基质细胞衰老对造血细胞衰老的影响  

Effects of senescent bone marrow stromal cells on aging of hematopoietic cells

作　　者：陈雄斌[1] 陈粼波[1] 刘颖[1] 景鹏伟 侯吉颖 夏婕妤 宋小英[1] 熊丽溶 王璐[1] 王亚平[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与胚胎学教研室,重庆400016

出　　处：《解剖学报》2016年第6期750-755,共6页Acta Anatomica Sinica

基　　金：国家自然科学基金(81173398;81673748);重庆市科委基础与前沿研究项目(cstc2014jcyjA 10001)

摘　　要：目的探讨骨髓基质细胞(BMSCs)衰老对骨髓造血细胞衰老的影响及其可能的机制。方法贴壁培养大鼠骨髓基质细胞,传代BMSCs至P3代时分组。对照组:常规培养;衰老组:常规培养基础上加入D-半乳糖(D-Gal)诱导BMSCs衰老,两组细胞分别培养48h。收集培养上清液,ELISA法检测细胞因子:粒细胞巨噬细胞集落刺激因子(GM-CSF)、干细胞因子(SCF)、白细胞介素(IL)-6、IL-1β,IL-2和肿瘤坏死因子-α(TNF-α)含量,CCK-8检测细胞增殖能力,衰老相关β-半乳糖苷酶(SA-β-Gal)染色检测BMSCs衰老。分离提取正常骨髓单个核细胞(BMMNCs),在衰老组与对照组BMMSCs上种植正常BMMNCs共培养24h,收集上层悬浮BMMNCs。CCK-8法测定细胞增殖能力;多向造血祖细胞集落(CFU-Mix)半固体培养法检测细胞增殖及分化能力;SA-β-Gal染色观察衰老细胞百分率;流式细胞术分析细胞周期与细胞凋亡;DCFH-DA荧光染色检测细胞活性氧簇(ROS)水平;酶学法检测细胞内丙二醛(MDA)含量和总超氧化物歧化酶(SOD)活性。结果衰老组BMSCs增殖能力显著下降,SA-β-Gal染色阳性细胞百分率增加,细胞分泌GM-CSF、SCF、IL-6、IL-1β水平明显降低,IL-2、TNF-α水平显著升高。与衰老BMSCs共培养后,BMMNCs的增殖能力显著下降,SA-β-Gal染色阳性细胞百分率显著上升,BMMNCs形成CFUMix集落数量明显降低,细胞周期呈现阻滞且细胞凋亡率上升,细胞内ROS、MDA含量上升,SOD含量下降。结论本实验中D-Gal成功复制了BMSCs衰老,衰老的BMSCs可诱导骨髓造血细胞衰老,其机制可能与BMSCs导致造血细胞氧化损伤有关。Objective To investigate the effect of senescent bone marrow stromal cells（ BMSCs） on aging of hematopoietic cells. Methods The BMSCs were isolated by whole bone marrow adherent culture from the rat,and passaged to 3rd generation（ P3）. The P3 generation of BMSCs was collected and divided into two groups. The control group was cultured as routine and the aging group was cultured with additional 30 g / L D-Galactose（ D-Gal） for 48 hours.And then,the amounts of granulocyte macrophage colony-stimulating factor（ GM-CSF）,stem cell factor（ SCF）,interfeukin（ IL）-6,IL-1β,IL-2 and tumor necrosis factor-α（ TNF-α） in BMSCs culture supernatant were assayed by ELISA; the proliferation ability of BMSCs was detected by cell counting Kit-8（ CCK-8）; the senescence-associated β-galactosidase（ SA-β-Gal） staining was used to detect the senescent BMSCs. Bone marrow mononuclear cells（ BMMNCs） from rat femur marrow were isolated and purified. Co-culturing aging BMSCs with BMMNCs for 24 hours,and then BMMNCs was collected. The proliferation ability of BMMNCs were measured by CCK-8; the proliferation and differentiation ability of CFU-Mix was detected by colony forming assay; the SA-β-Gal staining was used to detect the senescent BMMNCs; the cell cycle distribution and the ratio of apoptosis of BMMNCs were analyzed by flow cytometry（ FCM）; DCFH-DA fluorescentstaining and FCM were analyzed the level of reactive oxygen species（ ROS） in BMMNCs. Malondialdehyde（ MDA）content and total superoxide dismutase（ SOD） activity were analyzed using enzymatic assay. Results The proliferation of aging group BMSCs was significantly decreased. The positive ratio of SA-β-Gal was markedly increased. The amount of GM-CSF,SCF,IL-6,IL-1β in BMSCs culture supernatant of aging group were obviously decreased and the level of IL-2、TNF-α were raised. After co-cultured BMMNCs with aged BMSCs for 24 hours,the positive ratio of SA-β-Gal stained BMMNCs was significantly increased; the proliferation abil

关 键 词：骨髓造血细胞 骨髓基质细胞 衰老生物学 酶联免疫吸附测定法 大鼠 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]