顺铂诱导大鼠星形胶质细胞衰老的研究

CDDP induces cellular senescence of rat astrocytes in vitro

目的:探讨顺铂(CDDP)对星形胶质细胞增殖的影响,明确CDDP在星形胶质细胞衰老中的作用。方法:分离、纯化大鼠星形胶质细胞,分别应用0,1,5,10,20μmol/L的CDDP处理细胞24 h,再用新鲜培养液培养3 d,随后进行细胞活力检测。10μmol/L CDDP处理星形胶质细胞24 h,更换为新鲜培养液继续培养3 d后,分别应用免疫荧光检测Ki67阳性细胞,衰老相关β-半乳糖苷酶(SA-β-Gal)标记衰老细胞,Western Blot检测p16和p21的表达变化,并用流式细胞术检测细胞周期。结果:10μmol/L和20μmol/L CDDP处理星形胶质细胞24 h后,细胞活力明显下调;即使在新鲜培养液中继续培养3 d,细胞活力仍受明显抑制。10μmol/L CDDP减少Ki67阳性细胞的比例,但增加SA-β-Gal阳性细胞的比例;此外,10μmol/L CDDP还能够上调p16、p21的表达水平,并使细胞周期阻滞于S期。结论:CDDP能够抑制大鼠星形胶质细胞的增殖并诱导细胞衰老。

Objective： To investigate the effects of cis-Diaminodichloroplatinum（ CDDP） on astrocytes proliferation and cellular senescence,clarify CDDP role in astrocyte senescence. Methods： Rat astrocytes were isolated and purified,the cells were treated with 0,1,5,10,20 μmol/L CDDP for 24 h,then the cells were cultured in fresh medium.3 d later,the cell viability was detected. Moreover,the cells were cultured at 10 μmol/L CDDP for 24 h,then all cells were cultured in fresh medium for another 3 d. Then the immunocytochemistry was used to detect Ki67 positive cells,the senescence-associated-β-galactosidase（ SA-β-Gal） staining was used to label the senescent cells,the expression of p16 and p21 was measured by Western Blot,and the cell cycle was investigated by flow cytometry. Results： After treatment of 10 μmol/L and 20 μmol/L CDDP for 24 h,the cell viability was inhibited; after recovery in fresh medium for another 3 d,the cell viability was still inhibited. Moreover,10 μmol/L CDDP decreased the Ki67 positive cells proportion,but increased the SA-β-Gal-labeled senescent cells proportion; 10 μmol/L CDDP also upregulated the expression of p16 and p21,and arrested the cell cycle at S-phase. Conclusion： CDDP could inhibit the rat astrocytes proliferation and induce the cellular senescence.