用改良消减杂交筛选类风湿性关节炎滑膜细胞中高表达基因

 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

Screening of High-expressed Genes in Fibroblast|like Synovial Cells of Rheumatoid Arthritis Patients by an Improved Subtractive Hybridization

In order to study the mechanism of hyperplasy and invasiveness of fibroblast|like synovial cells(FLS)of rheumatoid arthritis(RA)patients,an improved subtractive hybridization was used to screen for the genes which expression was increased in RA FLS compared with FLS of osteoarthritis (OA) patients.The cDNA fragments of high|expressed genes obtained by subtractive hybridization were cloned into plasmid vectors and the false positive fragments were removed by reverse dot|blotting hybridization.Then the true positive fragments were sequenced and the sequences were compared with those genes in GenBank to identify whether they were known genes or novel sequences.At last,Northern|blotting hybridization was done to detect the expression level of some high|expressed genes in several RA and OA patients FLS cells.The results showed that,among the 150 cDNA fragments obtained after subtractive hybridization,36.7%(55/150)were longer than 400 bp,and about 8%(12/150) were longer than 1000 bp.among the 55 fragments longer than 400 bp,false positive rate was about 23.7%(13/55).Among the 18 fragments sequenced,12 were cDNA fragments of known genes including serine protease(IGF|1 binding protein specific,IGFBP),Cathepsin B and Laminin receptor,and other six were novel sequences,two of which were separately homologous to Ring|box protein 1 and SON DNA|binding protein.Northern|blotting hybridization results showed that IGF|1 binding protein specific serine protease,Cathepsin B and Laminin receptor could all be expressed in both RA and OA cells,but relatively more highly expressed in RA FLS cells.The results suggest that the improved subtractive hybridization is a simple and effective method to screen differentially expressed genes,and IGF|1 binding protein specific serine protease,Cathepsin B and Laminin receptor might be related to the hyperplasy and invasiveness of FLS in RA patients.