HLA-DM基因与类风湿关节炎的相关性研究

To investigate the association between HLA-DM alleles and rheumatoid arthritis, the effect of DM alleles on the susceptibility and pathogenesis of RA in Chinese population, and to quantify the levels of different cytokines in the supernatant of HLA-DR4 transfected Hela cells to understand the interaction between DR and DM in future . Methods： Ninety-six RA patients and 100 matched control subjects were genotyped in the third exon of HLA-DM gene by polymerase chain reaction/sequence specific oligonucleotide probe(PCR/SSOP). The HLA-DRB1*0405 positive individuals were screened by PCR/restriction fragment length polymorphism(PCR/RFLP). HLA-DMA,DMB and DRB transcripts in purified peripheral blood B cells with anti-CD19 mAb-coated magnetic beads from 36 RA patients were determined by semiquantitative and competitive RT-PCR. The HLA-DM and -DR protein expression were also quantified by Western blot. DRA DRB cDNA was cloned into a eukaryotic expression vector and transfected into Hela cells introduced by lipofectamine. The supernatant was collected and the cytokines were quantified by ELISA kits. Results: No significant difference in the distribution of the DMA and DMB alleles was observed between RA patients and controls , as well as those between the RA patients with HLA-DRB1*0405 and controls. The patients with DMB*0101 gene had a higher frequencies of HLA-DRB1*0405 and a higher RF titer than those with DMB*0102 allele(P<0.01 or 0.05) . Compared with normal controls or AS patients, only the transcription of HLA-DMA and DMB from RA patients was significantly decreased(P<0.01), but no that of HLA-DRB(P>0.05). An mean DM:DR ratio in RA patients was significantly lower than those in normal controls (P<0.01). The mean ratio of DMA/βactin rather than DRA/βactin in RA patients were dramatically decreased compared with that in normal controls at protein level(P<0.01). TNF-α、IL-2 and IFN-γ were significantly increased since the 2nd day after culture and reached the highest level at the 3rd or the 4th day and kept a relatively high level during a week of culture. Conclusion：HLA-DM polymorphisms may not have effect on the susceptibility of RA. However, specific HLA-DM down-regulation might play an important role in the processes of antigen presentation and autoimmune in RA. The DMB*0101 allele may play some important roles in the disease severity and activity of RA. HLA-DR4 gene might involve in the onset and progress of rheumatoid arthritis (RA) through the upregulation of Th1 cytokines.

采用PCR-RFLP、原位杂交、细胞培养、分子克隆、测序及表达等方法，分析DR4（+）RA患者的HLA-DRB1、DM基因多态性、mRNA和蛋白质表达，了解DM分子对DRB1和DR4分子的影响，探諨M在DRB1对RA易感性中的可能作用。并构建DR4（+）HeLa细胞系，获得大量DR4分子，为今后制备DR4单抗和DR4（+）DM（+）HeLa细胞系，进一步研究DM 和DR4的相互作用奠定基础。