寻常型天疱疮患者血清对HaCaT细胞Dsg、MMP-9表达的影响

 目的:探讨寻常型天疱疮（PV）患者血清对HaCaT细胞桥粒芯蛋白（Dsg）1、3和基质金属蛋白酶（MMP）9表达的影响及其参与PV棘层松解的可能机制。方法:用含5%PV血清的高糖DMEM培养基孵育HaCaT细胞，以正常培养组、健康血清组以及抗Dsg3抗体组（阳性对照组）作为对照。刮取细胞提取总RNA及蛋白质，Q PCR检测Dsg1、Dsg3和MMP 9 mRNA转录水平，Western Blot检测Dsg1、Dsg3和MMP 9表达，荧光单克隆Dsg1/3抗体分别检测细胞表面Dsg1、Dsg3表达。 结果:Q PCR结果显示：与正常培养组比较，含5% PV血清培养组HaCaT细胞Dsg1、Dsg3、MMP 9基因的mRNA相对表达量分别上升约432%、402%、402%，而抗Dsg3抗体组分别上升约495%、488%、604%，PV血清组与正常培养组间有明显差异（P值均<0.05）。 Western Blot结果显示：与正常培养组相比，PV血清组HaCaT细胞表面Dsg1、Dsg3、MMP 9蛋白表达升高。Dsg1、Dsg3荧光单克隆抗体检测显示：PV血清组HaCaT表面Dsg1线状荧光消失，替代为颗粒状、团块状荧光颗粒分散于细胞表面和胞浆区域，而细胞表面Dsg3线状荧光仍然存在，但强度减弱。 结论:PV血清能促进Dsg1、Dsg3和MMP 9转录及表达，Dsg3抗体可通过诱导角质形成细胞Dsg1蛋白内吞，削弱抗体补偿作用参与棘层松解。

Effects of pemphigus vulgaris serum on expression of Dsg and MMP-9 in HaCaT cells

Objective：To investigate the effect of pemphigus vulgaris (PV) serum on expression of Desmoglein (Dsg)1, Dsg3 and matrix metalloproteinase 9 (MMP) 9 in HaCaT cells, and its pathogenic role in acantholysis.  Method: This study included four experimental groups, i.e., normal control group, healthy serum group, PV serum group, anti Dsg3 monoclonal antibody group (positive group). Human HaCaT cells were cultured with high glucose DMEM medium containing 5% PV serum, followed by extraction of RNA and protein for Q PCR and Western Blot analyses, respectively. In addition, immunostaining was used to assess expression of Dsg1, Dsg3 and MMP 9 in keratinocytes. Results: In addition to elevations in expression levels of respective protein, culture of HaCaT cells with 5% PV serum increased expression levels of mRNA for Dsg1, Dsg3 and MMP 9 by 432%, 402% and 402%, respectively. Likewise, incubation of HaCaT cells with anti Dsg3 monoclonal antibody increased expression levels of mRNA for Dsg1, Dsg3 and MMP 9 by 495%, 488% and 604%, respectively (P<0.05 vs. normal control). Immunostaining showed that linear distribution of Dsg1 on cell surface was replaced with granular and clustered patterns on the cell surface and in the cytoplasm in keratinocytes cultured with PV serum. Moreover, intensity of Dsg3 staining was decreased. Conclusions: PV serum increases expression levels of Dsg1, Dsg3 and MMP 9. The Dsg3 antibody can cause acantholysis by induction of endocytosis of Dsg1 protein by keratinocytes and reduction in antibody compensation.