Expression of human α-galactosidase leads to reduction of major xenoepitope Galα(1,3)Gal in NIH 3T3 cell  

人α-半乳糖苷酶的表达抑制NIH 3T3细胞主要异种抗原表位Galα(1,3)Gal的生成(英文)

作　　者：闫景莲[1] 余路阳[1] 朱丽华[1] 郭礼和[1] 

机构地区：[1]中国科学院上海生命科学研究院生物化学与细胞生物学研究所

出　　处：《Acta Pharmacologica Sinica》2003年第10期985-990,1060,1061,共8页中国药理学报（英文版）

基　　金：Project supported by National Natural Science Fundation of China (39993430).

摘　　要：AIM: To examine the effects of the expression of α-galactosidase on the expression of the major xenoepitope Gal α(1,3) Gal (G antigen) in NIH 3T3 cell. METHODS: The expression levels of G antigen and H antigen and binding of human natural antibodies (IgG and IgM) and complement (C3c) to NIH 3T3 cells were analyzed by flow cytometry. Western blot was employed to further determine the expression of glycoproteins of G antigen. Cytolysis assay with normal human serum was performed by MTT assay. RESULTS: In transfectants, Western blot showed that the binding of human IgG to glycosylated proteins located on the cell membrane was decreased, even abrogated totally. Together with the reduced binding of Gs-IB4 (Griffonia simplicifolia) to transfectants, the stable expression of human α-galactosidase effectively inhibited Galα(1,3) Gal, Gal epitope synthesis in NIH 3T3 cell. As a result, the xenoreactivities of human IgG, IgM, and C3c were reduced by 73.4%, 22.3% and 47.9%, respectively, while the cell lysis mediated by uman XNA and complements was decreased by 42.4%. CONCLUSION: The stable expression of human α-galactosidase in NIH 3T3 cell strongly inhibits the expression of Gal epitopes, resulting in abrupt reduction in xenorejection induced by human serum.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。

关 键 词：human α-galactosidase Gal epitope XENOTRANSPLANTATION NIH 3T3 cell 

分 类 号：R392[医药卫生—免疫学]