不同年龄大鼠血清对骨髓间充质干细胞衰老影响的实验研究

The Study of Effects of Different Aged Serum on Mesenchymal Stem Cells Aging

制备成年(8～12周龄)和老年(64～72周龄)SD大鼠血清,实验随机分为两组:年轻血清组和老年血清组,分别采用成年和老年SD大鼠血清培养成年MSCs 36小时后,衰老相关β-半乳糖苷酶和活性氧染色观察细胞衰老,MTT法检测细胞增殖,AO/EB法和Hoechst 33342染色法观察细胞凋亡及存活情况。免疫细胞化学和Western blot法检测衰老相关蛋白γ-H2A.X、p53表达,RT-PCR法观察p53、p21 mRNA表达。结果发现,与年轻血清组相比,老年血清组MSCs衰老细胞数明显增加((96.2±24.1)/500细胞vS(30.8±8.2)/500细胞,P<0.01)、增殖能力减弱,凋亡率升高,γ-H2A.X、p53蛋白表达水平升高,p53、p21 mRNA表达升高。这些结果说明、老年SD大鼠血清可促进MSCs发生衰老变化,并抑制MSCs增殖及存活能力,这一作用可能与DNA损伤反应和p53/p21信号通路有关。

Rat serum was extracted from young （8-12 weeks） and aged （64-72 weeks） SD rats. SD rats were randomly divided into two groups, young serum and old serum groups. Adult MSCs were cultured with 10% young rat or old rat serum for 36 h. Senescence-associated changes were examined with SA-β-galactosidase staining and ROS staining. The proliferation ability was detected by MTT assays. The survived and apoptotic cells were determined by AO/EB staining and Hoechst 33342 staining. To further explored the mechanisms of old rat serum on the MSC aging, we detected the expression of γ-H2A.X, a molecular marker of DNA damage response, p53, and p21 by reverse transcription-PCR, immunofluorescence and Western blot. The results showed that the expression of γ-H2A.X, p53 and p21 was increased in the senescent MSCs induced by culture with old rat serum. Taken together, this study indicates that old rat serum can induce the senescence of MSCs and inhibits the proliferation and survival ability of MSCs, and suggests that the DNA damage response and the p53/p21 pathways may be the two main mediators of old rat serum induced MSCs aging.