衰老大鼠模型骨髓基质细胞的生物学特点

Characteristics of bone marrow stromal cells biology in aging rats model

目的探讨衰老大鼠骨髓基质细胞(BMSCs)的生物学特点,为阐释机体衰老对造血诱导微环境的影响提供实验依据。方法雄性健康SD大鼠随机分为正常组和衰老模型组。衰老模型组:大鼠皮下注射D-半乳糖120mg/kg,qd×42;正常对照组:大鼠皮下注射等时与等量生理盐水。衰老动物复制完成后第2天,分离骨髓单个核细胞(BMNCs)进行髓系造血祖细胞混合集落生成单位(CFU-Mix)培养。采用全骨髓贴壁法培养和传代BMSCs,取第3代细胞进行检测,CCK-8法测定BMSCs增殖能力;流式细胞术分析细胞周期;衰老相关β-半乳糖苷酶(SA-β-Gal)染色观察衰老BMSCs百分率;ELISA检测细胞培养上清液中白细胞介素(IL)-6、干细胞生长因子(SCF)含量;DCFH-DA荧光染色流式检测BMSC活性氧簇(ROS)水平;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;Western blotting检测衰老相关蛋白P16、P21、P53表达。结果与对照组相比衰老模型组大鼠CFU-Mix集落形成数量明显降低;BMSCs增殖能力显著下降;处于G0/G1期的BMSCs比例增高、S期细胞比例降低,细胞阻滞于G1期;SA-β-Gal染色阳性的BMSCs百分率显著上升;BMSCs培养上清液中IL-6、SCF含量明显下降;BMSC内ROS、MDA氧化损伤指标上升,SOD抗氧化指标下降;衰老相关蛋白P16、P21、P53表达明显上调。结论衰老大鼠骨髓基质细胞表现衰老相关生物学改变,其机制可能与氧化损伤激活衰老信号通路有关。

Objective To explore the characteristic of bone marrow stromal cells （BMSCs） biology in aging rats and to provide the theoretic and experimental evidences for explaining the effect of senescence on hematopoietic inductive microenvironment （HIM）. Methods Healthy male SD rats were randomized into two groups. The aging model rats were given 120mg D-galactose （D-Gal） by daily neck subcutaneous injection for 42 consecutive days. As a control, rats were administrated with the same volume of saline for the same period. On the second day after the aging model was established, the bone marrow mononuclear cells （BMNCs） were extracted from the femur bone marrow and cultured for CFU-Mix colony forming assay. The BMSCs were isolated by whole bone marrow adherent culture, and passaged to 3rd generation （F3） as well. For the F3 generation BMSCs, the ability of proliferation was detected by Cell Counting Kit-8 （CCK-8）; the distribution of cell cycle was analyzed by flow cytometry （FCM）; the senescence associated-β-galactosidase（SA-β-Gal） staining was used to detect the senescent BMSCs; the amount of interleukin（IL）-6 and stem cell factor （SCF） in BMSCs culture supernatant were detected by ELISA ; DCFH-DA fluorescent staining and FCM analyzed the level of reactive oxygen species（ROS） in BMSCs ; malonaldehyde（MDA） content and total superoxide dismutase （SOD） activity were analyzed as well using enzymatic assay; Western blotting examined the expression level of senescence-related proteins including P16, P21and P53. Results Compared with the control group, the capability of mixed colony forming unit （CFU-Mix） of BMNCs in aging model group was obviously attenuated. The results indicated that BMSCs of aging model rats displayed a decrease in proliferation; the BMSCs were held in G1 phase arrest, the proportion of the cells in G_0/G_1 phase increased, while the proportion in S phase decreased; the positive ratio of SA-β-Gal stained BMSCs also significantly increased; the amount of IL-6 and SCF in BMSCs culture supernatant of aging model group was lower than that of control. Furthermore the BMSCs in aging model rats showed an increase in ROS and MDA level and a decline in total SOD activity. The expression of senescence-related proteins including P16, P21 and P53 were obviously up-regulated. Conclusion BMSCs in aging model rats show senescence-associated biologic changes, and the underlying mechanism may be related to the activation of senescence signaling pathway due to oxidative damage.