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为了研究类风湿性关节炎 (rheumatoid arthritis,RA)滑膜细胞 (fibroblast- like synovialcells,FLS)过度增殖和破坏软骨的分子机理 ,利用改良消减杂交法以骨性关节炎 (osteoarthritis,OA)病人滑膜细胞为对照 ,筛选 RA滑膜细胞中的高表达基因 .将得到的基因片段克隆入质粒载体 ,通过反向点杂交排除假阳性克隆后 ,将阳性克隆进行核酸序列分析 ,最后用 Northern杂交方法检测一些高表达基因在 RA和 OA病人滑膜细胞中的表达水平 .结果显示 ,共分离到 1 50个 RA高表达基因片段 ,其中长于 1 0 0 0 bp的片段占 8% (1 2 /1 50 ) ,长于 40 0 bp的片段占 36.7% (55/1 50 ) ,在大于 40 0 bp的片段中 ,假阳性率为 2 3.7% (1 3/55) .在测序的 1 8个片段中 ,已知基因有 1 2个 ,其中包括 IGF- 1结合蛋白 (IGFBP)特异性丝氨酸蛋白酶、层粘连蛋白受体和组织蛋白酶 B等 .新序列有 6个 ,其中两个序列分别与 Ring- box蛋白 1和 SON DNA结合蛋白同源 .对 IGFBP特异性丝氨酸蛋白酶、层粘连蛋白受体和组织蛋白酶 B基因的 Northern杂交分析显示 ,在 RA病人滑膜细胞中 ,这些基因的表达水平高于 OA病人滑膜细胞 .这些结果提示 ,这种改良消减杂交法是一种简便有效的分离差异表达基因的方法 ;IGF- 1结合蛋白特异性丝氨酸蛋白酶、层粘
In order to study the molecular mechanism of over proliferation and destruction of cartilage in rheumatoid arthritis (RA) synovial cells (FLS), we used modified subtraction hybridization to treat patients with osteoarthritis (OA) Membrane cells as a control to screen high expression genes in synovial cells of RA.The cloned gene fragments were cloned into the plasmid vector and the false positive clones were excluded by reverse dot blot hybridization.The positive clones were subjected to nucleic acid sequence analysis and finally Northern blotting The expression of some highly expressed genes in synovial cells of patients with RA and OA was detected.The results showed that a total of 150 highly expressed RA gene fragments were found, of which 10% 50), 36.7% (55/150) longer than 40 bp, and false positive rate was 23.7% (1 3/55) in more than 40 bp. In the 18 fragments sequenced There are 12 known genes, including IGFBP-specific serine protease, laminin receptor and cathepsin B. There are 6 new sequences, two of which are related to Ring-box Protein 1 and SON DNA Syncytial homology.Expression of IGFBP-specific serine protease, laminin receptor and cathepsin B genes by Northern blot analysis showed that the expression level of these genes in synovial cells of RA patients was higher than that of OA patient synovial cells. The results suggest that this improved subtractive hybridization method is a simple and effective method for the isolation of differentially expressed genes; IGF-1 binding protein-specific serine protease, layer sticky