D-半乳糖诱导大鼠骨髓基质细胞衰老及其机制

目的构建大鼠骨髓基质细胞(BMSCs)体外和体内衰老模型,观察BMSCs衰老生物学特性。方法体外对照组:常规培养大鼠骨髓BMSCs,取第三代(P3)细胞继续培养48 h;体外衰老组:在对照组基础上加入D-半乳糖(D-Gal,终浓度30 g/L),作用48 h;体内衰老组:大鼠皮下注射D-Gal(120 mg/kg·d),qd×42 d;体内对照组:注射等时等量0.9%氯化钠溶液,模型完成第2天,分离培养BMSCs,取P3细胞进行实验。检测:CCK-8测定细胞增殖;流式细胞术分析周期和凋亡率;β-半乳糖苷酶(SA-β-Gal)染色观察BMSCs衰老百分率;DCFH-DA荧光流式细胞术检测活性氧簇(ROS)水平,酶学法检测过氧化物丙二醛(MDA)含量和总超氧化物歧化酶(SOD)活性;Western blot检测P16、P21、P53、CDK2和cyclin D表达。结果 D-Gal体外与体内致衰老组BMSCs增殖能力下降;细胞G0/G1期比例增高、S期比例降低(P0.05);SA-β-Gal染色阳性百分率上升(P0.05);胞内ROS、MDA上升,SOD下降(P0.05);P16、P21、P53表达上调,CDK2、cyclin D下调(P0.05)。结论 D-Gal在体内与体外均能构建BMSCs衰老模型,其机制与D-Gal诱导BMSCs氧化损伤和激活衰老信号途径相关。

Senescence induced by D-galactose and its biological mechanism in rat bone marrow stromal cells

Objective To establish an aging model of rat bone marrow stromal cells (BMSCs) in vitro and in vivo, in order to study the senescence biology of aging BMSCs .Methods The control cell group ( in vitro):isolating, puri-fying and culturing BMSCs from healthy male SD rats .collecting the third generation ( P3) of BMSCs for analysis . The aging model group (in vitro):the P3 BMSCs were incubated with D-Galactose (D-Gal, 30 g/L) for 48 hours. The aging rat model group ( in vivo): the rats were given 120 mg D-Gal by the way of daily neck subcutaneous injection for 42 consecutive days .The control rat group ( in vivo):the rats were administrated with the same volume of saline for the same times .On the second day after the aging model was established , the BMSCs were collecting and culturing for study.1)The proliferative potency was detected by cell counting Kit-8(CCK-8);the distribution of cell cycle and apoptosis was detected by flow cytometry (FCM);2)the ratio of aging BMSCs was examined by the senescence-associated β-Galactosidase(SA-β-Gal) staining;3)malonaldehyde(MDA) content and total super-oxide dismutase(SOD) was examined activity by enzymatic assay; the level of reactive oxygen species (ROS) by DCFH-DA fluorescent staining was counted with FCM;4 ) the expression level of senescence-related signaling was proteins of P16 , P21 , P53 , CDK2 and cyclin D by Western blot .Results Compared with the matched control group, the BMSCs of aging model group displayed a significant decrease in proliferation; the BMSCs were held in G1 phase arrest as the proportion of the cells in G 1 phase increased , while that decreased in S phase ( P<0.05 );and the positive ratio of SA-β-Gal stained BMSCs also significantly increased ( P <0.05 ); BMSCs in the aging model group showed an increasing level of ROS and MDA , meanwhile a decline in total SOD activity was decreased (P<0.05);P16,P21 and P53 protein expression in aging BMSCs was obviously enhanced (P<0.05), at the same time the expression of CDK2 and cyclin D was also decreased ( P<0.05 ) .Conclusions D-Gal can be used to develope an aging model of BMSCs .It acts through up-regulation of expressions of aging-related proteins and in-hibition of oxidative stress injury and chronic inflammation .