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。最近的研究表明，MCTD的PH是由内皮细胞损伤引起的。为了揭示MCTD患者PH的致病机制，本研究重点研究了抗内皮细胞抗体（anti-endothelial cell antibodies， AECA）及AECA靶蛋白的鉴定。采用细胞酶联免疫吸附法（cell - elisa）检测MCTD合并PH患者的AECA，采用微血管内皮细胞（HMVEC-L）检测。为了确定AECA的患病率，高于健康对照平均值2 sd的值被认为是阳性的。9例MCTD患者中有6例阳性结果。为了鉴定AECA的靶点，将HMVEC-L蛋白通过双向电泳分离并转移到PVDF膜上。对健康对照或MCTD患者的血清进行Western blot分析。选取不同样品中存在差异的检测蛋白点，通过肽质量指纹图谱进行鉴定。检测到约20个蛋白点，其中一半被确定。通过二维western blot分析，获得候选抗原。其中包括在各种疾病中作为自身抗原的蛋白质。此外，一种候选药物被认为与SSc患者的PH有关。这增加了鉴定蛋白中包含AECA抗原的可能性，表明二维western blot分析是检测AECA靶抗原的有效技术。

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.