ASSOCIATION OF GLUCOCORTICOID RECEPTOR β EXPRESSION AND STEROID INSENSITIVITY IN PATIENTS WITH AUTOIMMUNE BULLOUS DISEASES

人糖皮质激素受体前体基因的选择性剪接产生两种高度同源的异构体，保留的hGRα和hGRβ。与hGRα不同的是，hGRβ不结合糖皮质激素，在转录上是不活跃的。近年来，由于致死性哮喘和糖皮质激素耐药溃疡性结肠炎患者外周血单个核细胞中hGRβ的表达增加，hGRβ被认为在糖皮质激素抵抗中起一定作用。我们先前的研究表明，正常人外周血单核细胞中hGRβ的表达明显低于hGRα的表达。本研究的目的是阐明hGRα和hGRβ分析是否可以预测自身免疫性大疱病患者对糖皮质激素的反应。对5例天疱疮或大疱性类天疱疮患者外周血单个核细胞和7例健康志愿者外周血单个核细胞的HGR、α和β进行半定量RT-PCR一步法分析。半定量逆转录聚合酶链式反应显示，BP患者和健康志愿者外周血单核细胞中hGRβ的表达均显著低于hGRα的表达。然而，与半定量逆转录聚合酶链式反应的结果相比，实时定量聚合酶链式反应并不能显示hGRα和hGRβ表达水平的定量差异。用不同浓度的hGR-α和hGR-β特异性引物进行的第二次实验表明，hGR-α和hGR-β表达的相对比例可以随引物浓度的不同而变化。第三个实验使用了另一个新的引物和针对hGRα和hGRβ的TaqMan探针，也未能建立定量分析hGRα和hGRβ的最佳条件。

Alternative splicing of the human glucocorticoid receptor (hGR) pre-mRNA generates two highly homologous isoforms, reserved hGR α and hGR β. In contrast to hGR α, hGRβ does not bind glucocorticoids and is transcriptionally inactive. Recently, hGRβ was suggested to play a role in glucocorticoid resistance because the expression of hGR β is increased in peripheral blood mononuclear cells (PBMCs) in patients with fatal asthma and glucocorticoid-resistant ulcerative colitis. Our previous study demonstrate that the expression of hGR β is significantly lower tha that of hGR α in PBMC from normal volunteers as revealed by semiquantitative RT-PCR and immunohistochemistry. The aim of this study was to clarify whether analysis of hGR α and hGR β can predict the response to glucocorticoids in patients with autoimmune bullous diseases. Total RNA obtained from peripheral blood mononuclear cells (PBMCs) of 5 patients with pemphigus or bullous pemphigoid and 7 healthy volunteers was analysed by semiquantitative RT-PCR and one-step RT-PCR using specific TaqMan probe for hGR α and β. Semiquantitative RT-PCR revealed that the expression of hGR β is significantly lower than that of hGR α in PBMC both in patients with BP and healthy volunteers. However, in contrast to the results obtained from semiquantitative RT-PCR, the real time PCR could not demonstrate quantitative difference of expression level of both hGR α and hGR β. The second experiment with various concentration of primers specific for both hGR α and hGR β showed that relative ratio of hGR α and hGR β mRNA expression could fluctuate according to the primer concentration. The third experiment using another new primers and a TaqMan probe specific for hGR α and hGR β also failed to establish the optimal conditions for quantitative analysis of hGR α and hGR β.