论文部分内容阅读
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±10.63vs.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 glomerular mesangial cell lines with different expression levels of glucocorticoid receptor β (GRβ) and to study the effect of GRβ on the glucocorticoid effect of mesangial cells. Methods: The retroviral vector pLXSN was used to transfer the sense and antisense recombinant GRβ into mesangial cells. The cell lines were identified by cell culture, reverse transcription polymerase chain reaction, gene sequencing and Western blot analysis. MTT assay and flow cytometry were used to detect the effect of GRβ on the proliferation inhibition and cell cycle of dexamethasone. Results: The constructed glomerular mesangial cells had the correct integration of GRβ sense and antisense genes respectively. The expression of GRβ protein in glomerular mesangial cells transfected with GRβ sense gene was significantly higher than that of the GRβ antisense gene (109.74 ± 10.63vs.19.08 ± 1.01, P <0.05). The inhibitory effect of dexamethasone on the proliferation of glomerular mesangial cells with GRβ expression was significantly lower than that of the glomerular mesangial cells with low GRβ expression (18.47% ± 2.12% vs.60.33% ± 5.29%, P <0.05). Under the inhibition of dexamethasone, the cells transfected with the gene of sense GRβ gene were significantly lower in S phase cells and in G1 phase cells than those in untransfected cells. CONCLUSION: GRβ, which is highly expressed in glomerular mesangial cells, has the effect of antagonizing glucocorticoid effect. The intracellular expression of GRβ is an important factor that determines the sensitivity or resistance of cells to hormone.