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目的:检测人α-半乳糖苷酶在NIH3T3细胞系中对Gal抗原表位生成的抑制作用。方法:流式细胞术比较G抗原、H抗原和人天然抗体(IgG和IgM)的表达水平,Western blot检测G抗原糖蛋白的表达。四唑盐(MTT)比色试验检测NIH3T3细胞在人血清介导的裂解作用下的活力。结果:克隆了人α-半乳糖苷酶并在NIH3T3细胞上建立了永久细胞系,Western blot结果表明,在表达人α-半乳糖苷酶的NIH3T3细胞中,人的天然抗体与细胞表面糖蛋白的结合能力显著降低,甚至完全消失,同时凝集素Gs-IB4结合分析也进一步证明人α半乳糖苷酶的稳定表达能有效地抑制NIH3T3细胞上Gal抗原表位的生成,流式细胞检测结果则表明,由Gal抗原表位与人IgG,IgM和3C3c介导的异种反应性分别降低了73.4％,22.3％和47.9％,随后发生的细胞裂解反应也被显著抑制。结论:人α-半乳糖苷酶能够有效抑制鼠成纤维细胞NIH 3T3细胞表面Gal抗原表位的生成以及Gal抗原表位与人天然抗体共同介导的免疫排斥。
OBJECTIVE: To examine the inhibitory effect of human α-galactosidase on Gal epitope production in NIH3T3 cell line. Methods: The expression of G antigen, H antigen and human natural antibody (IgG and IgM) were compared by flow cytometry. The expression of G antigen glycoprotein was detected by Western blot. Tetrazolium salt (MTT) colorimetric assay was used to examine the viability of NIH3T3 cells under human serum-mediated lysis. RESULTS: Human α-galactosidase was cloned and a permanent cell line was established on NIH3T3 cells. The result of Western blot showed that in human NIH3T3 cells expressing human α-galactosidase, human natural antibodies bind to cell surface glycoprotein Of the binding capacity of GFP was significantly reduced, or even disappeared completely, and agglutinin Gs-IB4 binding analysis further demonstrated that the stable expression of human α-galactosidase can effectively inhibit the generation of Gal epitopes on NIH3T3 cells. The results of flow cytometry Showed that the xenogenic reactivity mediated by Gal epitopes with human IgG, IgM, and 3C3c decreased by 73.4%, 22.3% and 47.9%, respectively, and subsequent cell lysis reactions were also significantly suppressed. CONCLUSION: Human α-galactosidase can effectively inhibit the generation of Gal epitopes on the surface of NIH 3T3 cells and the immunological rejection mediated by Gal epitopes and human natural antibodies.