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

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）作用下，产生以纤连蛋白（Fibronection,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AngⅡ致细胞外基质堆积作用的相关基因，为探讨AngⅡ在肾小球硬化发展过程的分子机制奠定基础。方法：人MsC经AngⅡ（10^-6mol/L)刺激24h后，采用抑制性消减杂交（Suppression subtrative hybridization,SSH)获得AngⅡ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Norhern筛选上调表达的基因cDNA片段，并以Northern杂交验证，然后进行120个克隆，经反向Northern筛选上调的基因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：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，泛素蛋白连接酶等，3个克隆（克隆104，52，46）与已知序列没有明显同源性，提示为新基因，3个新基因分别命名为AngRem(AngiotensinⅡ related gene in mesangial cells)104,AngRem52,AngRem46,GenBank登录号分别为AF367870，YA040225，AY040224，结论：对已知基因的分析表明，Ang II对细胞外基质具有双重作用。即：既刺激培养的人MsC细胞外基质成分表达上调，也通过刺激PAI－I等分子的表达而制抑细胞外基质成分的降解，此外，我们还发现了目前尚未报告的与AngⅡ对MsC的生物学效应－细胞外基质堆积和增生等细胞相关的基因（包括3个新基因），为揭示AngⅡ介导的MsC在肾小球硬化进展过程的可能分子机制提供了新的线索。

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

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.