大鼠肾小球系膜细胞表型的增龄性变化

The characteristic and significance of rat mesangial cells with advancing age

目的观察大鼠肾小球系膜细胞随增龄细胞表型及功能的变化特点。方法取3、12、24月龄健康雄性Wistar大鼠原代系膜细胞进行培养,MTT法检测细胞增殖能力,进行SA-β-gal染色,采用RT-PCR和Western印迹法分别检测p21WAF1/CIP1/SDI1基因及蛋白质的表达变化,并用免疫荧光法检测系膜细胞中p21WAF1/CIP1/SDI1的表达与定位。结果大鼠肾小球系膜细胞增殖能力随增龄逐渐减弱(P<0.05),SA-β-gal染色阳性率随增龄逐渐增加(P<0.05),p21WAF1/CIP1/SDI1mRNA及蛋白质表达随增龄逐渐增加(P<0.05)。免疫荧光结果示p21WAF1/CIP1/SDI1主要在细胞核内表达,荧光强度随增龄逐渐增强(P<0.05)。结论大鼠肾小球系膜细胞随增龄细胞形态、增殖能力与衰老相关蛋白表达发生明显改变,出现了复制性衰老表型。

Objective To investigate the characteristic of rat mesangial cells with advancing age. Methods Male Wistar rats aged of 3, 12, 24 months were used. The rat mesangial cells were processed for MTT assay and senescence associated β-galactosidase （SA-β-gal） staining. The expression and location of p21^WAF1/CIP1/SDI1 in mesangial cells were studied by immunofluorescence. The mRNA and protein levels of p21^WAF1/CIP1/SDI1 were detected by Western blot and/or RT-PCR, respectively. Results The ability of proliferation was decreased in rat mesangial cells with advancing age（ P 〈 0. 05 ）. The expression of senescence associated β-galactosidase activity was increased in mesangial cells with advancing age. Percentages of SA-β-gal staining positive cells were （ 11.9 ± 3.6 ） % versus （ 39. 0 ± 4. 0 ） % versus （ 86. 9 ± 7.4） % （P 〈0.05） in young, middle and aging glomerular mesangial cells. The p21^WAF1/CIP1/SDI1 protein showed localization in nucleus in mesangial cells, and the staining intensity of p21^WAF1/CIP1/SDI1in mesangial cells in old animals was markedly increased, as compared to that in young and middle age rats （ P 〈 0.05 ）. The mRNA levels of p21^WAF1/CIP1/SDI1 was significantly increased in mesangial cells in old animals, as compared to that at the age of 3 and 12 months, meanwhile the p21^WAF1/CIP1/SDI1 proteins expression were dramatically increased with advancing age （ P 〈 0. 05 ）. Conclusions The cellular phenotype and function are changed in rat mesangial cells with advancing age. The rat mesangial cells appear replicative senescence with advancing age.