血管紧张素Ⅱ上调人肾小球系膜细胞硬化相关基因的克隆

Screening and identification of the up-regulated genes in human mesangial cells induced by angiotensin Ⅱ

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。

Objective To explore the possible molecular mechanism of angiotensin II(AngII)on human mesangial cells (MsC) . Methods Suppression subtractive hybridization (SSH) was performed to screen and identify the over-expressed genes during the extracellular matric accumulation in MsC, which were stimulated by Ang II. cDNAs from human MsC without and with Ang II -stimulated for 24 hours were synthesized and SSH was performed. All subtracted cDNAs were subcloned into pGEM-Teasy Vector and 120 clones were randomly picked up for differentially screening by using reverse Northern blot. The over-expressed clones were sequenced by automated DNA sequence analyzer and homology search was performed by NCBI on-line sever. Finally, the over-expressed genes were confirmed by Northern blot analyses. The full-length sequences of novel genes were obtained by RACE and long distance-PCR. Results Among 120 randomly picked clones, .55 clones that over-expressed in human MsC were selected by reverse Northern blot. 18 over 55 clones that increased in expression of Ang II-induced MsC were further confirmed by Northern blot analyses. Sequence analysis and homology search of these clones revealed that 15 clones completely metchedwith known genes in GenBank, including thrombspondin-1, collagen I (a2), plasminogen activator inhibitor-1, etc. There were three novel and nucharacterized genes. Furthermore, the full length cDNAs of three novel genes were amplified by RACE-PCR. AngRem104(GenBank accession No: AF0367870)contains 1665bp nucleartides and coding a 347aa peptide. AngRem 52(GenBank accession No: AY040225) contains 3151bp nucleartides and coding a 340aa peptide. AngRem46( GenBank accession No: AY040224) contains 388bp nucleartides and coding a 60aa peptide. Conclusions Ang II has dual effects on the extracellular matrix accumulation in human mesangial cells. It not only improves the expression of ECM components but also inhibits the degradation of ECM by up-regulate special molecules. The further investigation of all identified known and unknown genes may give new insight into the molecular mechanism related to the role of Ang II in the progression of chronic renal disease.