Lin Guiting, Chow Sylvia, Lin Jackie, Wang Guifang, Lue Tom F, Lin Ching-Shwun
Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, California 94143-1695, USA.
J Cell Biochem. 2004 May 1;92(1):104-12. doi: 10.1002/jcb.20043.
It has been shown that rat aortic smooth muscle cells (AoSMCs) lost PKG-I expression when propagated repetitively or grown at low densities. Conversely, AoSMCs isolated from PKG-I deficient mice are indistinguishable from those isolated from normal mice in morphology and growth characteristics. In this study, human AoSMCs were grown from passage 9 (p9) to passage 15 (p15) and rat AoSMCs were isolated and cultured from p1 through p15. Western blotting and immunofluorescence microscopy showed little difference in PKG-I expression among different passages. Next, rat AoSMCs of p4 were grown and harvested at different cell densities. Western blotting again showed little difference among cells seeded or harvested at different densities. To test the effect of cell passage on PKG-I activation, rat AoSMCs of p4 and p11 were treated with cGMP and analyzed by Western blotting for phosphorylated vasodilator-stimulated phosphoprotein (P-VASP). The results showed that p4 had higher level of PKG-I activation than p11.
研究表明，大鼠主动脉平滑肌细胞（AoSMCs）在反复传代或低密度培养时会失去PKG-I表达。相反，从PKG-I缺陷小鼠分离的AoSMCs在形态和生长特性上与从正常小鼠分离的AoSMCs没有区别。在本研究中，人AoSMCs从第9代（p9）培养至第15代（p15），大鼠AoSMCs从第1代（p1）分离并培养至第15代。蛋白质印迹法和免疫荧光显微镜检查显示不同传代之间PKG-I表达差异不大。接下来，将第4代（p4）大鼠AoSMCs以不同细胞密度进行培养和收获。蛋白质印迹法再次显示，不同密度接种或收获的细胞之间差异不大。为了测试细胞传代对PKG-I激活的影响，用cGMP处理第4代（p4）和第11代（p11）大鼠AoSMCs，并通过蛋白质印迹法分析磷酸化血管舒张刺激磷蛋白（P-VASP）。结果显示，第4代（p4）的PKG-I激活水平高于第11代（p11）。
