GRβ对肾小球系膜细胞糖皮质激素效应的影响

摘要

目的:构建糖皮质激素受体β(GRβ)不同表达水平的肾小球系膜细胞株,研究GRβ对肾小球系膜细胞糖皮质激素效应的影响.方法:利用逆转录病毒载体pLXSN将重组GRβ正义和反义基因转入肾小球系膜细胞,经细胞培养、逆转录聚合酶链反应、基因测序和蛋白质印迹分析鉴定所构建的细胞株,应用MTT法和流式细胞术检测细胞内不同表达水平的GRβ对地塞米松的细胞增殖抑制效应和细胞周期变化的影响.结果:构建的肾小球系膜细胞分别有正确的GRβ正义和反义基因整合,转染了GRβ正义基因的肾小球系膜细胞内GRβ蛋白质表达水平明显高于转染了GRβ反义基因者(109.74±10.63 vs.19.08±1.01,P＜0.05);地塞米松对GRβ高表达的肾小球系膜细胞的增殖抑制效率显著低于GRβ低表达的肾小球系膜细胞(18.47%±2.12% vs.60.33%±5.29%,P＜0.05).在地塞米松的抑制下,转染了正义GRβ基因的细胞中,其S期细胞降低程度和G1期细胞升高的程度均明显低于未转染的细胞.结论:肾小球系膜细胞内高表达的GRβ具有拮抗糖皮质激素效应的作用,细胞内GRβ表达水平是决定细胞对激素敏感或抵抗的重要因素.%Objective To construct mesangial cell lines over- or under- expressing glucocorticoid receptor β ( GRβ ) , to investigate the effect of GRβ on glucocorticoid biological function, and to determine whether the overexpression of GRβ explains the glucocorticoid-resistant in glomerular mesangial cells (GMCs). Methods The recombinant human sense or anti-sense gene of GRβ was transferred into the rat GMCs by retrovirus-mediated stable transfection technique. Expression of hGRβ mRNA in GMCs was determined by reverse transcription of total RNA followed by quantitative reverse transcriptionpolymerase chain reaction (RT-PCR) , and the product of RT-PCR was then analyzed by gene sequencing. The expression of hGRβ protein in GMCs was tested by Western blot. The inhibitory rate of dexamethasone-mediated cells on lipolysaccharide (LPS)-stimulated GMC proliferation was detected to assess the effect of GRβ at different expression levels on the glucocorticoid action. The cell proliferative activity in different cells with different levels of GRβ was tested by MTT chromatometry. The change of cell cycle was analyzed by flow cytometry. Results RT-PCR and gene sequencing showed that the recombinant sense and anti-sense genes were correctly integrated into genomic DNA of mesangial cells.The protein expression tested by Western blot showed that GRβ in cells inserted with the sense hGRβ gene was higher than those cells inserted with the anti-sense hGRβ gene (109. 74 ± 10. 63 vs.19.08 ± 1.01 , P ＜ 0.05 ). The inhibitory rate of cell proliferation induced by dexamethasone was lower in GMCs transfected with sense hGRβ gene than those transfected with anti-sense hGRβ gene ( 18.47 % ± 2. 12 % vs. 60.33 % ± 5.29 % , P ＜ 0.05 ). Under the inhibition of dexamethasone,the decreased cell number of S-stage cells was significantly lower, and the increased cell number of G1-stage cells was significantly higher in GMCs transfected with sense hGRβ gene than those of non-transfected cells. Conclusion The overexpression of GRβ may inhibit the glucocorticoid action in GMCs.The GRβ level in mesangial cells may be an important factor in determining whether they are sensitive or resistant to glucocorticoid.