【摘要】： 背景與目的：蛋白酶活化受體-1(PAR-1)是一種的蛋白質(zhì)，由425個(gè)氨基酸組成，含7個(gè)疏水跨膜域，為典型的G蛋白偶聯(lián)受體。PAR-1廣泛分布在各種組織細(xì)胞上，包括上皮細(xì)胞，血小板，內(nèi)皮細(xì)胞，成纖維細(xì)胞，單核細(xì)胞，T淋巴細(xì)胞，成骨細(xì)胞，平滑肌細(xì)胞，神經(jīng)細(xì)胞，神經(jīng)膠質(zhì)細(xì)胞及某些癌細(xì)胞系等。PAR-1具有廣泛的生物學(xué)效應(yīng)，參與諸多疾病的發(fā)生發(fā)展，因此，，我們制備廉價(jià)、有效的抗PAR-1mAb具有重要意義。 方法：以PAR-1特異性片段為免疫原(13肽)免疫BALB／c小鼠，取免疫小鼠脾細(xì)胞和小鼠骨髓瘤NS-1細(xì)胞融合。通過間接ELISA法篩選可特異性分泌抗PAR-1 mAb的雜交瘤細(xì)胞。采用capture ELISA法鑒定mAb的Ig亞類；通過ELISA、Dot blot、免疫組織化學(xué)染色法、流式細(xì)胞分析、激光共聚焦掃描顯微鏡技術(shù)鑒定mAb的特異性。 結(jié)果：獲得2株可穩(wěn)定分泌抗PAR-1 mAb的雜交瘤細(xì)胞，其亞型分別為IgM，IgG2b。Dot blot檢驗(yàn)表明，mAbs可與膜上的抗原結(jié)合。免疫組織化學(xué)染色表明，mAb與人扁桃體、結(jié)腸組織中的淋巴細(xì)胞、包皮組織中的成纖維細(xì)胞、肺組織中的巨噬細(xì)胞反應(yīng)呈陽性。流式細(xì)胞儀分析顯示，2株mAb與人肺腺癌細(xì)胞系A(chǔ)549細(xì)胞膜上及細(xì)胞胞漿內(nèi)的受體均呈陽性反應(yīng)。激光共聚焦掃描顯微鏡觀察，A549細(xì)胞的胞膜及胞漿內(nèi)均有熒光標(biāo)記的陽性反應(yīng)物。 結(jié)論：成功地制備抗PAR-1 mAb，為變態(tài)反應(yīng)性疾病，炎癥性疾病及其他疾病的研究工作提供了有用的試劑。
[Abstract]:Background & objective: protease activated receptor-1 (PAR-1) is a kind of protein consisting of 425 amino acids and contains 7 hydrophobic transmembrane domains. PAR-1 is a typical G-protein-coupled receptor. PAR-1 is widely distributed in various tissues and cells. Including epithelial cells, platelets, endothelial cells, fibroblasts, monocytes, T lymphocytes, osteoblasts, smooth muscle cells, nerve cells, PAR-1 has a wide range of biological effects and participates in the occurrence and development of many diseases. Therefore, it is of great significance to prepare cheap and effective anti-PAR-1mAb. Methods: BALB/c mice were immunized with PAR-1 specific fragment (13 peptide). Spleen cells of mice were fused with NS-1 cells of myeloma mice. Hybridoma cells secreting anti-PAR-1 mAb were screened by indirect ELISA method. The Ig subclasses of mAb were identified by capture ELISA method, and the specificity of mAb was identified by ELISA,Dot blot, immunohistochemical staining, flow cytometry and laser confocal scanning microscopy. Results: two hybridoma cells stably secreting anti PAR-1 mAb were obtained. The subtypes of these hybridomas were IgM,IgG2b.Dot blot test. The results showed that mAbs could bind to the antigens on the membrane. Immunohistochemical staining showed that mAb reacted positively with lymphocytes in human tonsil, colon, fibroblasts in prepuce and macrophages in lung tissue. The results of flow cytometry showed that both mAb and A549 cells showed positive reaction to the receptors on the cell membrane and in the cytoplasm of A549 cell line. Fluorescence labeled positive reactants were observed in the membrane and cytoplasm of A549 cells by confocal laser scanning microscopy. Conclusion: the successful preparation of anti-PAR-1 mAb, may provide useful reagents for the research of allergic diseases, inflammatory diseases and other diseases.
【學(xué)位授予單位】：汕頭大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

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中國期刊全文數(shù)據(jù)庫 前1條

1 王海燕,何韶衡,鄭燕珊;蛋白酶激活受體1介導(dǎo)人肺上皮細(xì)胞分泌MCP-1[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2005年17期

本文編號(hào)：2381124