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目的 筛选和鉴定培养的人肾小球系膜细胞（MsC）在血管紧张素Ⅱ（Angiotensin Ⅱ,Ang Ⅱ）作用下,产生以纤连蛋白（Fibronectin,FN）为代表的细胞外基质成分的过程中上调表达的基因,寻找Ang Ⅱ致细胞外基质堆积作用的相关基因,为探讨Ang Ⅱ在肾小球硬化发展过程的分子机制奠定基础。方法 人MsC经Ang Ⅱ（10-6mol/L）刺激24h后,采用抑制性消减杂交（suppressionsubtractive hybridization,SSH）获得Ang Ⅱ相关的差异表达的cDNA,经纯化克隆到pGEM-Teasy Vector并转化大肠杆菌;随机选择120个克隆,经反向Northern筛选上调表达的基因cDNA片段,并以Northern杂交验证,然后进行DNA序列测定和同源性比较。采用5＇-和3＇-RACE及长距离PCR的方法获得新基因的全长cDNA。结果 在120个随机选择的克隆中,反向Northern结果表明有55个克隆表达明显上调。挑选其中20个克隆进行DNA序列测定,结果显示18个为独立的基因片段序列（有两个序列为双拷贝）,其中15个为已知基因,包括细胞外基质成分:如血小板反应素Ⅰ、Ⅰ型胶原α2;细胞骨架及结合蛋白:如平滑肌肌动蛋白α、钙调蛋白1、γ-胞浆型肌动蛋白等;合成和代谢相关蛋白:如醛缩酶A、延长因子1-γ、arnesyl pyrophosphate synthetase等;蛋白分解相关蛋白:如组织蛋白酶、泛素
Objective To screen and identify the process of cultured human glomerular mesangial cells (MsC) producing extracellular matrix components such as fibronectin (FN) under the action of Angiotensin Ⅱ (Ang Ⅱ) Upregulation of genes to find Ang Ⅱ induced extracellular matrix accumulation related genes, to explore the Ang Ⅱ in the development of glomerular sclerosis molecular mechanism of the foundation. Methods Human MsC was induced by Ang Ⅱ (10-6mol / L) for 24 hours, and the differentially expressed cDNA of Ang Ⅱ was obtained by suppressionsubtractive hybridization (SSH). The cDNA was purified and cloned into pGEM-Teasy Vector and transformed into E.coli ; 120 clones were randomly selected and the cDNA fragments of the up-regulated genes were screened by reverse Northern blot and verified by Northern blotting. DNA sequencing and homology comparison were performed. The full-length cDNA of the new gene was obtained by 5’- and 3’-RACE and long-distance PCR. Results Of 120 randomly selected clones, reverse Northern results showed that 55 clones were significantly up-regulated. Twenty of these clones were selected for DNA sequencing. The results showed that 18 were independent gene fragment sequences (two copies were double copies), of which 15 were known genes including extracellular matrix components such as platelet-responsive factor I, Type I collagen α2; cytoskeleton and binding proteins: such as smooth muscle actin α, calmodulin 1, γ-cytoplasmic actin; synthesis and metabolism-related proteins: such as aldolase A, elongation factor 1-γ , Arnesyl pyrophosphate synthetase, etc .; protein degradation related proteins: such as cathepsin, ubiquitin