肾上腺髓质素对大鼠肝星状细胞表达细胞周期蛋白的影响

The effect of adrenomedullin on the expression of cell cycle proteins in hepatic stellate cells-T6 in vitro

目的探讨肾上腺髓质素(ADM)对活化肝星状细胞(HSC)表达细胞周期蛋白的影响。方法采用不同浓度的ADM对HSC-T6细胞进行干预12h、24h和48h。采用MTT法检测HSC-T6细胞增殖;采用SABC法检测细胞增殖核抗原(PCNA)、细胞周期蛋白D1(cyclin D1)和细胞周期素依赖性激酶抑制因子P21waf1的表达。结果在ADM10-7mol/L浓度下作用12h,24h,48h,细胞生长抑制率逐渐增加(分别是7.88%,17.58%,18.79%);在ADM10-7mol/L作用24h时,HSC的PCNA、cyclin D1和P21waf1表达的平均光密度分别为0.387±0.023、0.454±0.029和0.508±0.011,而对照组分别为0.246±0.030、0.209±0.014和0.401±0.033(t值分别为5.149、5.752和5.299,P<0.05),差异有显著性。相关性分析表明,PCNA和cyclinD1的变化呈正相关趋势(r=0.834,P<0.05),而PCNA和P21waf1呈负相关趋势(r=-0.770,P<0.05),cyclinD1和P21waf1呈负相关趋势(r=-0.796,P<0.05)。结论肾上腺髓质素可抑制HSC-T6细胞增殖。ADM作用HSC可能通过调控HSC的细胞周期,抑制PCNA和cyclinD1表达,促进P21waf1表达,从而发挥抑制HSC增殖作用。

Objective To investigate the effect of adrenomedullin （ADM） on expression of cell cycle proteins in hepatic stellate cells-T6 in vitro. Methods The proliferation of hepatic stellate cells-T6 was evaluated by MTT assay and the expression of proliferating cell nuclear antigen（PCNA）,cyclin Dland P21^wafl protein was examined by streptavidin-biotin complex at 12h,24h and 48h culture in different concentrations of ADM. Results The rates of HSC proliferation at 12h, 24h and 48h were inhibited with ADM at 10^-7 mol/L by 7. 88% ,17.58% and 18.79% ,respectively. The expression of PCNA and cyclin D1 protein in the HSC treated with ADM at the concentration of 10^-7 mol/L for 24h were significantly reduced as compared with those in control groups （the average optical density were 0. 387 ± 0. 023 versus 0. 246 ± 0. 030, P d0.05 and 0. 45±0. 029 versus 0. 209±0. 014,P〈0.05,respectively）. The P21wafl protein was markedly increased as compared with in control （the average optical density was 0. 508 ± 0. 011 versus 0. 401 ± 0. 033, P〈0.05 ）. The expression of PCNA was significantly positively correlated with cyclin D1 expression（P〈0. 05） and an significantly negative correlation were found between the expression of PCNA and P21^wafl （P〈0.05）,and between cyclin D1 and P21^wafl （P〈0.05）. Conclusion ADM can inhibit the proliferation of HSCs-T6,which might through inhibiting the expression of PCNA and cyclin D1 and stimulating P21^wafl expression in HSC-T6 cells.