视神经脊髓炎患者水通道蛋白4基因单核苷酸多态性位点的研究

摘要

目的：从基因水平研究水通道蛋白4（AQP4）基因外显子区域单核苷酸多态性（SNP）位点以及其在视神经脊髓炎发病机制中的可能作用。方法选取2010年3月—2012年6月湖北医药学院附属襄阳医院收治的视神经脊髓炎患者72例为视神经脊髓炎组，同时期本院收治的多发性硬化患者80例为多发性硬化组，取全血 DNA 进行AQP4基因的SNP位点分析，并通过特异性的定点诱变技术对携带 AQP4基因表达序列的绿色荧光蛋白表达载体（ pEGFP）重组质粒进行定点诱变，根据相应SNP位点突变生成相应突变质粒，脂质体转染法将相应质粒转染细胞建立抗AQP4抗体检测细胞株。分析视神经脊髓炎患者6种突变细胞株与原始细胞株血清抗AQP4抗体滴度水平及视神经脊髓炎组与多发性硬化组不同细胞株血清抗AQP4抗体滴度阳性率的差异。结果视神经脊髓炎组共发现6个AQP4基因SNP位点，位于2号和5号外显子，分别为R108T、I110N、E280R、D281R、P295R、E317M。1、3、4号外显子区域未发现AQP4基因SNP位点，多发性硬化组未发现相应的AQP4基因SNP位点。构建R108T、I110N、R108T/I110N、E280R/D281R、P295R、E317M细胞株，R108T/I110N、E280R/D281R、E317M细胞株与原始细胞株血清抗AQP4抗体滴度水平比较，差异有统计学意义（ p 〈0.05）。视神经脊髓炎组与多发性硬化组中原始细胞株及 R108T、I110N、R108T/I110N、E280R/D281R、P295R、E317M细胞株血清抗AQP4抗体滴度阳性率比较，差异均有统计学意义（ p〈0.05）。结论视神经脊髓炎患者AQP4基因SNP位点可能会导致表达蛋白一级结构发生变化，可以改变其抗原性，抗原抗体反应的强度不同可能是导致不同突变组间滴度差异的原因。%Objective To study the single nucleotide polymorphic ( SNP ) site in exon region of aquaporin ―4 (AQP4)gene and its possible effects on the pathogenesis of neuromyelitis optica(NMO)based on gene level. Methods 72 patients with NMO who were treated in Xiangyang Hospital Affiliated to Hubei University of Medicine from March 2010 to June 2012 were selected as NOM group,and 80 patients with multiple sclerosis admitted to this hospital during the same period were treated as multiple sclerosis group. The SNP site analysis of AQP4 was made to the whole blood DNA, and site ― directed mutagenesis was conducted to the recombinant plasmid of plasmid enhanced green florescence protein ( pEGFP ) that carried AQP4 gene expression sequence by specific site ― directed mutagenesis. The corresponding mutated plasmids were generated according to mutation of relevant SNP sites,and lipofection transfection method was used to build anti―AQP4 antibodies of the corresponding plasmid transfected cells so as to detect the cell line. The difference of serum antibody titers level of anti―AQP4 between six mutated cell lines and original cell lines in patients with NMO and positive rate of antibody titer of serum anti―AQP4 of different cell lines between NMO and multiple sclerosis groups were both analyzed. Results Six SNP sites of AQP4 gene were found in NMO group, which were located in exon 2 and 5, they were R108T, I110N, E280R, D281R, P295R and E317M. SNP sites of AQP4 gene were not found in exon 1,3 and 4,and no corresponding SNP sites of AQP4 gene was found in the multiple sclerosis group. Builded the R108T,I110N,R108T/I110N,E280R/D281R,P295R,E317M cell lines. There was significant difference in the antibody titer level of serum anti―AQP4 between the R108T/I110N,E280R/D281R,E317M and the original cell lines(p〈0. 05). There was significant difference in the positive rate of serum anti―AQP4 antibodies of the original cell lines and cell lines R108T,I110N,R108T/I110N,E280R/D281R,P295R,E317M between NMO group and multiple sclerosis group(p〈0. 05). Conclusion The SNP sites of AQP4 gene in patients with NMO may lead to changes in the primary structure of expressing protein and can change its antigenicity. The different intensity of the antigen―antibody reaction may be the reason of the difference in titers between mutation groups.