用差示PCR法筛选类风湿性关节炎滑膜细胞中的高表达基因_医学论文.docVIP

用差示PCR法筛选类风湿性关节炎滑膜细胞中的高表达基因_医学论文 用差示PCR法筛选类风湿性关节炎滑膜细胞中的高表达基因_医学论文 作者：王吉村　药立波　吕厚山　刘新平　赵锦荣　邓艳春　孙铁铮　聂晓燕 【关键词】 关节炎 　　关键词: 关节炎，类风湿滑膜细胞PCR 　　摘 要:目的 筛选类风湿性关节炎（RA）滑膜细胞中的高表达基因，以探讨RA滑膜细胞发生肿瘤样增殖和侵蚀软骨的分子机理. 方法 以骨性关节炎（osteoarthritis，OA）滑膜细胞作对照，采用差示PCR（differentially display PCR）方法，克隆RA滑膜细胞中高表达的cDNAs片段，经反向点杂交排除假阳性克隆后，将阳性克隆进行基因序列分析. 结果 共回收76个差异条带，得到42个有插段的克隆，经反向点杂交后得到22个阳性克隆，对其中19个克隆进行基因序列分析表明，最长片段为501bp长，最短片段为145bp长，大部分在300bp.有13个克隆为已知基因，其中包括组织蛋白酶，其它6个克隆为未知序列，这些未知序列中除一个序列外都为基因的非编码区. 结论 差示PCR方法在筛选差异表达基因方面存在着很大的局限性组织蛋白酶B可能与RA滑膜细胞的软骨侵蚀性有关. 　　Keywords:arthritis，rheumatoidsynovial cellsPCR 　　Abstract:AIM To simultaneously identify several genes whose expression increase in fibroblast-like synovial cells（FLS）of rheumatoid arthritis（RA）patients and to be relat-ed to the proliferation or invasion of FLS to cartilage of RA patients.METHODS Total RNAs were prepared separately from FLS of RA patients and osteoarthritis（OA，as control）patients.The RNA samples were analyzed by using differen-tially display reverse transcription polymerase chain reaction（DDRT-PCR）.Complementary DNA（cDNA）fragments corresponding to differentially expressed messenger RNAs（mRNAs）were eluted，cloned，and sequenced.The obtained sequences were compared with those genes which entered into EMBL/Genebank database.RESULTS Seventy-six differ-entially displayed fragments were obtained，but only55%（42/76）were successfully cloned into plasmid vectors.Re-verse dot-blotting hybridization showed that of the42clones，only22clones were truely positive.The sequence analysis of the19clones showed that，their lengths were from145bp to501bp，most of which were about300bp in length.13se-quences corresponded to known mRNAs，including cathepsin B.The other six sequences corresponded to transcripts of yet-unidentified genes.Most of the novel sequences were too short and were not in the coding regions of mRNAs.CON┐CLUSION DDRT-PCR method has many limitations on screening differentially expressed genescathepsin B might be related to the i