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

人糖皮质激素受体（human glucocorticoid receptor，hGR）前体mRNA的选择性剪接产生两种高度同源的异构体：保留型hGR α和保留型hGR β。与hGR α相反，hGR β不结合糖皮质激素，并且在转录上无活性。近年来，由于哮喘和糖皮质激素抵抗性溃疡性结肠炎患者外周血单个核细胞（PBMC）中hGR β的表达增加，hGR β被认为在糖皮质激素抵抗中发挥作用。我们前期的研究表明，正常人PBMC中hGR β的表达明显低于hGR α。本研究的目的是阐明分析hGR α和hGR β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。采用半定量RT-PCR和一步法RT-PCR方法，以特异性TaqMan探针检测5例天疱疮和大疱性类天疱疮患者及7例健康志愿者外周血单个核细胞（PBMCs）总RNA。半定量RT-PCR结果显示，BP患者和健康志愿者PBMC中hGR β的表达均显著低于hGR α。然而，与半定量RT-PCR的结果相反，真实的时间PCR不能显示hGR α和hGR β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 mRNA表达的相对比例随引物浓度的变化而波动。第三次实验采用另一种新的引物和hGR α和hGR β特异性TaqMan探针，也未能建立定量分析hGR α和hGR β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.