Research on the pathogenic mechanism of pulmonary hypertension (PH) in mixed connective tissue disease (MCTD).

混合性结缔组织病（MCTD）肺动脉高压（PH）的发病机制尚不清楚。近年来的研究表明，MCTD的PH是由内皮细胞损伤引起的。为揭示MCTD患者肺动脉高压的发病机制，本研究从抗内皮细胞抗体（anti-endothelial cell antibodies，AECA）和AECA靶蛋白的鉴定两方面入手，采用微血管内皮细胞（microvascularendothelial cell，HMVEC-L）为抗原，采用Cyto-ELISA检测MCTD肺动脉高压患者的AECA。为了确定AECA患病率，高于2 S.D.高于健康对照组平均值视为阳性。9例MCTD患者中有6例阳性结果。采用双向电泳技术分离HMVEC-L蛋白，并将其转移到PVDF膜上，以鉴定AECA的靶蛋白。用来自健康对照或MCTD患者的血清进行蛋白质印迹分析。通过肽质量指纹图谱分析，筛选出样品间存在差异的蛋白质点。共检测到约20个蛋白质点，其中半数蛋白质点已被鉴定，经双向免疫印迹分析，获得了候选抗原。这些包括在各种疾病中被报道为自身抗原的蛋白质。此外，其中一个候选者被认为与SSc患者的PH相关。这增加了鉴定的蛋白质包括AECA抗原的可能性，表明二维蛋白质印迹分析是用于检测AECA靶抗原的有用技术。

The pathogenesis of pulmonary hypertension (PH) in mixed connective tissue disease (MCTD) is still unclear. Recent studies have suggested that PH in MCTD is resulted from endothelial cell injury. To reveal the pathogenic mechanism of PH in patients with MCTD, this research was focused on anti-endothelial cell antibodies (AECA) and identification of target proteins of AECA.Cyto-ELISA was performed to detect AECA in MCTD patients with PH using micro vascular endothelial cell (HMVEC-L). To determine AECA prevalence, values higher than 2 S.D. above the mean of healthy control were considered as positive. Six of 9 MCTD patients had positive results. The titer of AECA was higher in MCTD patients with PH than without PH.For identification of targets of AECA, proteins from HMVEC-L were separated by two-dimensional electrophoresis and transferred on PVDF membranes. Western blot analyses were performed with sera from healthy control or MCTD patients. Detected protein spots with a difference among the samples were selected and identified by peptide mass finger printing. About 20 protein spots were detected and half of these were identified.As the results of two-dimensional western blot analyses, candidate antigens were obtained. These included proteins reported as autoantigen in various diseases. Furthermore, one of candidates is suggested to be associated with PH in patients with SSc. This raises possibility that the identified proteins include antigens of AECA, indicating that two-dimensional western blot analysis is useful technique for detection of the target antigens of AECA.