白癜风患者血清抗黑素细胞抗体的免疫荧光检测

Immunofluorescence test of serum antibody against melanocytes in vitiligo patients

目的建立抗黑素细胞抗体免疫荧光检测方法,测定白癜风患者血清中抗黑素细胞抗体。方法取外科手术后人眼睑正常皮肤,用胰蛋白酶消化后制成细胞悬液,在M2黑色素细胞选择性培养基中培养,每周传代1次,经4～5次传代,以免疫荧光和多巴胺染色检测培养细胞的纯度和生物学特性,转移到明胶包被的玻璃片上生长至70%融合,丙酮固定后,与不同稀释度的白癜风患者和正常对照血清反应,加入FITC标记的兔抗人抗体,观测荧光染色效果。结果黑素细胞能在培养基中选择性生长,经4～5w培养获得纯的黑素细胞,无成纤维细胞和角质形成细胞的污染。19例发展期患者抗黑素细胞抗体阳性15例,抗体阳性率79%;10例高于1:50,5例抗体滴度为1:10。稳定期15例中,抗黑素细胞抗体阳性4例,抗体阳性率27%。结论建立了抗黑素细胞抗体免疫荧光检测方法,白癜风患者血清中有抗黑素细胞抗体,且其滴度与病情发展相关。

Objective To establish a immunofluorescence method for the test of serum antibody against melanocytes in vitiligo patients. Methods Suspension of epidermal cells including melanocytes was made by trypsin digestion of normal epiderm obtained from eyelid operation. Then melanocytes were selectively cultured and proliferated in serum - free M2 medium. After 4 to 5 passages （ 1 passage per week ） and identification with immunological and biological examinations, the melanocytes were transferred to the glass slide coated with gelatin till 70% confluence, and then they were fixed with acetone, reacted with different dilution sera in vitiligo patients and normal controls. FITC labeled rabbit anti - human antibody was added to detect the fluorescence of melanocytes. Results Melonacytes could he selectively proliferated in the medium. After 4 to 5 passages, pure melanocytes without contamination of fibroblasts and keratinocytes were harvested. The antibodies against melanocytes were positive in 15 developing cases with the positive ratio of 79%. Among them,antibody titers of 10 cases were over 1：50 and 5 cases were 1：10. There were 4 of 15 stable cases with positive antibody against melanocytes and the positive ratio was 27%. Conclusion A immunofluorescence method for the test of serum antibody against melanoeytes in vitiligo patients is established successfully. The serum antibodies against melanocytes of vitiligo patients can be detected and its titers are related with the development of pathogenetic condition.