D-半乳糖对大鼠骨髓间充质干细胞衰老的影响

Effects of D-galactose on ageing of rat mesenchymal stem cells

目的:观察D-半乳糖(D-galactose,D-gal)对骨髓间充质干细胞(Mesenchymal Stem Cells,MSCs)衰老的影响,构建体外MSCs衰老诱导模型。方法:分离SD大鼠(7日龄)MSCs,传代培养至第3代后,细胞随机分为4组:分别为正常对照组,1 g/L,10 g/L和50 g/L D-gal衰老诱导组。对照组在正常的DMEM培养液中继续培养;诱导组MSCs分别在含1 g/L、10 g/L、50 g/L D-gal的DMEM培养液中培养48 h。各组细胞培养结束后,衰老相关β-半乳糖苷酶染色法观察细胞衰老变化、Western blot法检测衰老相关蛋白p53、p21和p16表达,AO/EB双染法检测细胞凋亡,MTT法检测细胞增殖,超氧化物歧化酶(SOD)活力和丙二醛(MDA)含量检测细胞内氧化应激水平。结果:10 g/L和50 g/L D-gal诱导组衰老相关β-半乳糖苷酶染色阳性细胞数较对照组显著升高(P<0.01),衰老相关蛋白p53、p21和p16表达明显增高:AO/EB染色结果显示50 g/L D-gal诱导组细胞凋亡率较对照组显著升高(P<0.01);MTT检测结果表明10 g/L和50 g/L D-gal均能抑制MSCs增殖活性,而10 g/L和50 g/L D-gal诱导组MSCs内SOD活力较对照组明显降低(P<0.01),MDA含量显著升高(P<0.01)。结论:10 g/L和50 g/L D-gal均可诱导MSCs发生衰老变化,并促使细胞内衰老相关蛋白p53、p21和p16表达增加,细胞存活能力减弱,增殖能力降低,这可能与D-gal促使MSCs内氧化应激水平升高有关。10 g/L是D-gal诱导MSCs衰老的合适浓度。

Objective： To investigate the effects of D-galactose （D-gal） on aging of rat marrow mesenchymal stem cells （MSCs） and its mechanism. Methods： MSCs isolated from young （7 d） SD rats were randomly divided into four groups：control group, 1 g/L, 10 g/L and 50 g/L D-gal treatment groups. In control group MSCs were cultured in DMEM containing 10% FBS for 48 h. In the D-gal treatment groups, MSCs were cultured in DMEM containing 10% FBS with 1 g/L, 10 g/L or 50 g/L D-gal for 48 h.The senescence-associated changes were examined with SA-β-galactosidase （SA-β-gal） staining, the expressions of p53, p21 and p16 were detected by Western blot. The aliving and apoptotic cells were determined by AO/EB staining. Cell proliferation was detected by MTT assay. SOD activity was measured by xanthine oxidase method,and the MDA content was estimated with thiobarbituric acid （TBA） method. Results： Compared to control group, the number of SA-β-gal positive cells and the expression of p53, p21 and p16 were significantly increased in the 10 g/L and 50 g/L D-gal treatment groups. The apoptosis rate in 50 g/L D-gal group was significantly higher than that in control group （P 〈 0.01 ）. The proliferation of MSCs was decreased in the 10 g/L and 50 g/L D-gal groups compared to control group （P 〈 0.05 ）. After 10 g/L and 50 g/L D-gal treatment, SOD activity was significantly decreased （P 〈 0.01 ）, and MDA level was increased （ P 〈 0.01 ）. Conclusion： The aging of MSCs can be induced by 10 g/L and 50 g/L D-gal,which may be associated with the elevated levels of oxidative stress.