摘要
目的探讨人类白细胞抗原(HLA)DRB1*0405小干扰RNA(small interfering RNA,siRNA)对类风湿关节炎(RA)相关的HLA-DRB1*0405蛋白表达的影响。方法构建HLA-DRB1*0405蛋白表达质粒siCHECKTM-2/HLA-DRB1*0405,同时针对HLA-DRB1*0405基因序列设计并构建6个小发夹RNA(short hairpin RNA,shRNA)表达质粒和1个针对无关序列的shRNA表达质粒。脂质体瞬时共转染两种质粒至人胚胎肾细胞293,以仅转染HLA-DRB1*0405蛋白表达质粒作为阴性对照,采用实时荧光定量PCR检测293细胞中HLA-DRB1*0405mRNA的表达,通过测定荧光素酶强度分析293细胞中HLA-DRB1*0405蛋白水平。结果所设计的6个HLA-DRB1*0405siRNA可不同程度的抑制HLA-DRB1*0405在293细胞中的表达,对mRNA水平,其抑制率最高可达89·25%,而在蛋白水平,其抑制率达46·70%。结论质粒介导的HLA-DRB1*0405siRNA可明显抑制HLA-DRB1*0405在人胚胎肾细胞株293中的表达,为利用RNA干扰(RNA interference,RNAi)抑制RA中HLA-DRB1介导的异常免疫反应提供了试验依据。
Objective To investigate the effects of small interfering RNA （siRNA） of HLA DRB1 ＊ 0405 HLA-DRB1 ＊ 0405 gene expression with plasmid-based siRNAs. Method Plasmid expressing HLADRB1＊ 0405- renilla fusion protein-siCHECK^TM-2/HLA-DRB1＊ 0405, 6 different short hairpin RNAs （shRNAs） targeting 6 19 bp nucleotide sequences of HLA-DRB1 ＊ 0405 （siRNA1 - 6）, and one shRNA targeting the control non-specific sequence （siRNAC） were designed and constructed. Human embryonic kidney cells of the line 293 were cultured and co-transfected by lipidosome with the plasmid siCHECK^TM-2/ HLA-DRB1＊ 0405 and one specific shRNA expressing vector transiently, and cells without shRNA- transfection were used as negative controls. The impact of RNAi on HLA-DRB1 ＊ 0405 expression was analyzed by real time fluorescence quantificational RT-PCR and luciferase test. Results The expression of HLA-DRB1 ＊0405 gene RNA of the 293 cells transfected with siRNA1, siRNA2, siRNA3, siRNA, and siRNA6 were down-regulated to 10.75%, 83.22%, 30. 63%, 48.54%, and 89.92% that of the control group with the inhibition rates of 89.25%, 16.78%, 69.37%, 51.46%, and 10.08% respectively. However, no significant downregulation was showed in the cells transfected with siRNA4 and siRNAC. The 293 cells transfected with siRNA1 and siRNA 3 showed a significant downregulation of the protein expression of HLA-DRB1 ＊ 0405 gene with the inhibitory rates of 6.70% and 36.85% respectively; however, the cells transfected with siRNA2, siRNA4, siRNAS, and siRNA6 did not show a significant downregulation. ConeJusion The significant inhibition of HLA-DRB1 ＊ 0405 gene expression by siRNA suggests a therapeutic approach in rheumatoid arthritis： to use RNA interference （RNAi） to inhibit the abnormal immune reaction mediated by HLA-DRB1 in rheumatoid arthritis.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2006年第11期732-735,共4页
National Medical Journal of China
关键词
RNA干扰
关节炎
转染
荧光素酶
RNA interference
Arthritis
Transfection
Luciferases
