去甲肾上腺素对原代培养的人肺动脉阻力血管平滑肌细胞蛋白表达谱的影响

Proteomic analysis of the effect of noradrenalin on human pulmonary arterial smooth muscle cells

目的用蛋白质组学方法分析去甲肾上腺素（NE）刺激人肺动脉阻力血管平滑肌细胞（PASMC）增殖前后细胞蛋白表达谱的变化。方法通过组织贴块法建立人PASMC细胞株；细胞经同步化后，用10-5 mol/L NE处理细胞24h，对照组未作处理，采用双向凝胶电泳、质谱和生物信息学蛋白质组方法比较NE处理前后细胞蛋白表达谱的变化，并用质谱分析差异在5倍以上的蛋白质分子；进一步使用实时RT-PCR和Western blot法验证差异蛋白的表达变化。结果 原代培养的人PASMC细胞纯度＞99％;PASMC经NE刺激后，多种细胞蛋白表达发生改变，其功能涉及细胞骨架相关蛋白、代谢及信号转导等多方面；实时RT-PCR和Western blot结果显示，NE刺激后在细胞内转录和翻译水平，α-烯醇化酶基因表达上调，与二维电泳结果一致。结论NE刺激人PASMC后，可引起细胞内多种不同功能的蛋白表达发生改变，并部分通过α-烯醇化酶基因调控细胞生物学功能。

ObjectiveTo observe the effect of noradrenalin（NE） on human pulmonary arterial smooth muscle cells （PASMC） by using a proteomic approach.Methods Human PASMC were cultured primarily in vitro.Experiments were performed in the 3rd to 5th passages of the cells.The human PASMC were cultured in serum-free medium for 24 h prior to treatment with either NE （10-5 mol/L, the test group）or completed-serum culture medium （the control group） for 24 h. And then analysis via 2-DE gel electrophoresis and MALDI-TOF-MS was performed to display the different protein profiles of whole cell protein from cultures of the control and the NE-treatment group. Real-time RT-PCR and Western blot analysis were used to confirm the proteomic analysis.Results The purity of the primary culture cells was about 99%.When the human PASMC were treated by NE, the expression of different groups of cellular proteins was changed, including cell cytoskeleton-associated proteins, cell signal-associated proteins, and glycolytic and metabolism-associated proteins. The results were confirmed using real-time RT-PCR and Western blot. NE enhanced the proliferation of human PASMC partly by affecting the expression of αenolase.Conclusion The data suggest that a wide range of signaling pathways may be involved in NEinduced proliferation of human PASMC, and α-enolase associated pathway may be an important one.