抗黑素细胞抗体免疫荧光细胞化学检测条件探讨

The study on conditions of immunofluoresence-cytochemistry test of autoantibodies to human melanocytes

目的建立一种简单、实用、准确的抗黑素细胞抗体免疫荧光细胞化学检测方法,用于白癜风患者血清抗黑素细胞抗体的检测及其相关抗原分析研究。方法青少年包皮组织经分离酶(Dispase)Ⅱ及胰酶消化后,获取细胞悬液,用M2黑素细胞培养基及Accutase细胞消化液选择及扩大培养,取第3代黑素细胞在培养皿中直接用冷甲醇固定(-20℃),白癜风患者及健康人血清用抗体稀释液按1:10比例稀释进行反应,洗涤后加入FITC标记的羊抗人抗体,封片后荧光显微镜下观察;取不同荧光强度的标本血清用Western-Blot法进一步验证检测。结果获得高纯度、高活性的黑素细胞,黑素细胞在培养皿中经冷甲醇直接固定后细胞形态保持完整,实验过程无脱落,与白癜风患者及健康人对照血清反应后在荧光显微镜下发出不同亮度荧光,强阳性血清荧光强度+++^++++,发光部位为黑素细胞胞质和胞膜;弱阳性血清发光较明亮,亮度+^++,发光部位主要集中在胞膜;阴性血清不发光,只见隐约细胞轮廓,相同血清经重复检测2次后结果相同;经Western-Blot检测后证实黑素细胞胞质和胞膜有多种蛋白与抗黑素细胞抗体阳性血清结合,蛋白染色带的部位及颜色深浅与免疫荧光法检测结果相一致。结论本文建立的抗黑素细胞抗体的免疫荧光细胞化学检测方法简单、准确、重复性好,可全面地反映体内抗黑素细胞抗体表达水平,适用于临床检测及相关抗原的基础研究。

Objective To establish a simple and accurate immunofluoresence-cytochemistry method of autoantibodiesto melanocytes. Methods The cell mixtures obtained from the foreskin tissues which were digested with dispaselland typesin separately were cultured in M2 melanocyte medium supplied with human melanocyte growth supplement, and then were subcultured by using Accutase. The third passage melanocyte were fixed with methanol stored at -20℃ and reacted with the sera of vitiligo patients and normal controls at 1：10 dilution, and then incubation with sheep anti-human IgG-FITC. All the sera were reconfirmed by performing Western-Blot. Results The melanocytes were selectively propagated in M2 melanocyte medium and the second subcultured cells obtained about 100% purity without contamination of fibroblasts and keratinocytes. The fixed cells maintained a good shape and without phenomina of detaching from the surface during testing. The fluorescence brightness was very different according to the level of antibodies to melanocytes, and the special antigens were located in the membrane and plasm of melanocytes or in membrane only. The similar results obtained after the assay described above were repeated two times with the same samples. The results were accurately confirmed by Western-Blot. Conclusion The method of immunofluoresence-cytochemistry established in this passage was very simple and accurate, it was suitable to detect the autoantibodies to melanocytes and the study of its related autoantigens.