D-半乳糖诱导大鼠骨髓基质细胞衰老  

D-galactose induces aging of rat bone marrow stromal cells

作　　者：姜蓉[1] 景鹏伟 宋小英[1,2] 熊丽溶 王亚平[1,2] 王璐[1,2] 

机构地区：[1]重庆医科大学干细胞与组织工程研究室,重庆400016 [2]重庆医科大学组织胚胎学教研室,重庆400016

出　　处：《中国组织化学与细胞化学杂志》2015年第6期512-516,共5页Chinese Journal of Histochemistry and Cytochemistry

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项目(cstc2014jcyj A10001)

摘　　要：目的建立体外骨髓基质细胞(bone marrow stromal cells,BMSSCs)衰老模型,分析衰老BMSCs生物学特性,为进一步研究衰老BMCs对造血细胞的影响奠定实验基础。方法雄性健康SD大鼠BMSCs全骨髓贴壁法体外培养,实验分为对照组和衰老模型组。对照组为常规培养48h;衰老模型组为常规培养基内加入D-半乳糖。CCK-8法测定BMSCs增殖能力;流式细胞术分析细胞周期;衰老特异性β-半乳糖苷酶(SA-β-Gal)染色观察衰老BMSCs百分率;酶学法检测BMSCs内过氧化物丙二醛(MDA)含量和总超氧化物歧化酶(SOD)活性,DCFH-DA流式荧光检测BMSCs活性氧簇(ROS)水平;Western Blotting检测BMSCs衰老相关信号蛋白P16、P21、P53的水平;ELISA检测BMSCs培养上清液中IL-1β、GM-CSF、SCF的含量。结果与对照组相比,衰老模型组BMSCs增殖能力显著下降;处于G1期的BMSCs比例增高、S期细胞比例降低,细胞阻滞于G1期;BMSCs SA-β-Gal染色阳性率显著上升;胞内ROS、MDA含量上升,SOD含量下降;P16、P21、P53水平明显上调;BMSCs培养上清液中IL-1β、GM-CSF、SCF含量明显下降。结论 D-半乳糖诱导骨髓基质细胞衰老可能与氧化损伤激活衰老相关信号通路有关,衰老骨髓基质细胞分泌活性造血生长因子减少。Objective To establish the aging model of bone marrow stromal cells( BMSCs) and analyze the biological property of aging BMSCs in vitro, thus to provide the experimental foundation for further exploring the effects of aging BMSCs on hematopoietic cells. Methods BMSCs were isolated from healthy male SD rats by whole bone marrow adherent culture, and divided into the control group and the aging model group. The control group was routinely cultured for 48 hours; the aging model group was cultured with additional D-galactose. The proliferation potency of BMSCs was detected by Cell Counting Kit-8( CCK-8); the distribution of cell cycle by flow cytometry( FCM); the ratio of senescent BMSCs by the senescence-associated β-galactosidase( SA-β-Gal) staining; MDA( malonaldehyde) content and total SOD( superoxide dismutase) activity by enzymatic assay; the level of ROS( reactive oxygen species) by DCFH-DA fluorescent staining counted with FCM; the expression level of senescence-related signaling proteins P16, P21 and P53 by Western Blotting; and the amount of IL-1β, GM-CSF and SCF in BMSC culture supernatant by ELISA. Results Compared with those in the control group, the BMSCs in the aging model group displayed a significant decrease in proliferation; the BMSCs were held in G1 phase arrest as the proportion of the cells in G1 phase increased while that in S phase decreased; and the positive ratio of SA-β-Gal stained BMSCs also significantly increased. The BMSCs in the aging model group showed an increase in ROS and MDA level and a decline in total SOD activity; P16, P21 and P53 protein expression in aging BMSCs was obviously enhanced; the amount of IL-1β, GMCSF and SCF in BMSC culture supernatant of the aging model group was lower than that of controls. Conclusion D-galactose induces bone marrow stromal cells aging, and the underlying mechanism may be related to the oxidative stress-induced activation of the senescence-related signaling pathway. Aging BMSCs secrete less active hematopoietic growth factors.

关 键 词：骨髓基质细胞 衰老 氧化应激 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]