【作者】 张翠华；

【导师】 王宗元； 栗占国；

【作者基本信息】 扬州大学 ， 临床兽医学， 2006， 硕士

【摘要】 类风湿关节炎(RA)的易感性与人类白细胞抗原(HLA)-DR1和HLA-DR4(均属HLA-DRB1)的等位基因亚型密切相关。国内外学者的研究表明，干扰RA相关性HLA-DRB1的作用可以抑制HLA-DRB1介导的异常免疫反应。RNA干扰(RNAi)是由双链RNA分子介导的序列特异性抑制同源基因表达的技术。引用RNAi机制沉默HLA-DRB1特定基因，可为RA的治疗提供新的思路。 由于我国、日本及韩国等亚洲国家RA患者主要与HLA-DRB1~*0405基因呈强相关性，研究抑制该基因的特异性小干扰RNA(small interfering RNA，siRNA)可以为我国RA患者的治疗提供试验依据。本研究设计了6条针对HLA-DRB1~*0405基因的shRNA和一条随机对照的shRNA，分别克隆到psiLentGene-2载体中。同时构建荧光素酶/HLA-DRB1~*0405重组质粒。用脂质体共转染两种重组质粒到人胚胎肾细胞(HEK-293)，通过实时荧光定量PCR和荧光素酶强度检测分析293细胞中HLA-DRB1~*0405基因mRNA的水平。 研究结果表明，设计的6条siRNA可不同程度抑制293细胞中HLA-DRB1~*0405基因的mRNA表达水平，实时荧光定量PCR的研究结果表明，其基因沉默效率最高可达89.25％，荧光素酶强度检测的结果表明，其基因沉默效率达46.70％，而对照组siRNA没有RNAi作用。根据结果筛选得到具有显著干扰作用的siRNA1和siRNA3。通过HLA-DR亚型分析，确立HLA-DRB1~*0405基因型RA患者。通过流式细胞术研究siRNA1和siRNA3对RA患者外周血单个核细胞(PBMC)表面HLA-DRB1~*0405基因表达的抑制作用。结果表明siRNA1试验组和siRNA3试验组的PBMC细胞HLA-DRB1~*0405蛋白表达阳性率分别为60.45％和54.67％，皆显著低于对照siRNA试验组的98.41％和非RNAi试验组的99.57％。更多还原

【Abstract】 The susceptibility of rheumatoid arthritis strongly related to the DRB1 allele subtypes of human leukocyte antigen. Many researches have demonstrated that the inhibition of DRB1 could avoid the abnormal immunoreaction. The significant inhibition by siRNA might suggest a therapeutic approach in rheumatoid arthritis.As people susceptive to in Asian RA are mostly born of DRB1*0405 gene, our research concentrated on siRNA aimed at HLA-DRB1*0405 gene in order to develop experiment basis for the new therapy. Plasmids to express renilla luciferase protein, 6 different shRNA targeting HLA-DRB1*0405 and a shRNA targeting the scramble sequence were designed and constructed. Human embryonic kidney cells 293 were co-transfected by lipidosome with psiCHECK-2/HLA-DRB1*0405 and one specific shRNA expression vector transiently, and cells without shRNA-transfection were used as the negative control. The impact of RNAi on HLA-DRB 1*0405 expression was analyzed by the real time fluorescence quantitative real-time PCR and the detection of renilla intensity.The research found that the mRNA of HLA-DRB 1*0405 gene in 293 cells was inhibited by siRNA, with a maximal inhibition rate of 89.25% detected by real-time fluorescence quantitative real-time PCR and 46.70% analyzed by the luciferase detection. According to the results, siRNA1 and siRNA3 were screened out with remarkable inhibition effect. We singled out RA patient with HLA-DRB 1*0405 allele subtype and separated peripheral blood mononuclear cells (PBMC) from blood. RNAi to DRB1*0405 gene on PBMC was studied and analyzed through Flow Cytometry. The positive rate of DRBl*0405 protein in the groups of siRNAl and siRNA3 is 60.45% and 54.67% separately, evidently lower than the scrambled siRNA group and the non-siRNA group.更多还原