Murray T R, Marshall B E, Macarak E J
Center for Research in Anesthesia, School of Medicine, University of Pennsylvania, Philadelphia 19104.
J Cell Physiol. 1990 Apr;143(1):26-38. doi: 10.1002/jcp.1041430105.
The use of cultured vascular smooth muscle cells for the study of events related to excitation and contraction of smooth muscle has been limited by the inability to reliably induce contractile responses after subculturing of the cells. This limitation has been overcome by the cell culture preparation described herein. We demonstrate that appropriate responses to both smooth muscle agonists and vasodilators were preserved in cells that were serially subcultured. Fetal bovine pulmonary artery and aortic cell cultures were established following enzymatic dispersion of the medial portion of freshly harvested vessels. At various times after isolation, cells were transferred to microscope coverslips coated with a polymerized silicone preparation (polydimethyl siloxane). Tension forces generated by the cells were manifested as wrinkles and distortions of this flexible growth surface. Visual evidence of cell contraction in the form of increased wrinkling was documented for cells exposed to angiotensin II, carbachol, and KCl. Decreases in cell tension occurred following treatment with isoproterenol, and those relaxing effects were overcome by subsequent treatment with the agonist carbachol. The contractile responses did not diminish with prolonged maintenance in culture or repeated subculturing. Phosphorylation of the light chains on the contractile protein myosin was also measured as a biochemical index of agonist-induced contraction. Cells depolarized with KCl or exposed to carbachol showed increased myosin phosphorylation when analyzed by 2-dimensional gel electrophoresis. The responses remained intact through 7 passages and 9 weeks in culture. These results show that cultured vascular smooth muscle cells do not necessarily undergo a phenotypic modulation with loss of contractility under prolonged maintenance in culture.
用于平滑肌兴奋和收缩相关事件研究的培养血管平滑肌细胞，一直受到细胞传代培养后无法可靠诱导收缩反应的限制。本文所述的细胞培养制备方法克服了这一限制。我们证明，在连续传代培养的细胞中，对平滑肌激动剂和血管扩张剂的适当反应得以保留。在对新鲜收获血管的中膜部分进行酶分散后，建立了胎牛肺动脉和主动脉细胞培养物。在分离后的不同时间，将细胞转移到涂有聚合硅氧烷制剂（聚二甲基硅氧烷）的显微镜盖玻片上。细胞产生的张力表现为这种柔性生长表面的皱纹和变形。对于暴露于血管紧张素II、卡巴胆碱和氯化钾的细胞，以皱纹增加形式出现的细胞收缩的视觉证据得到了记录。用异丙肾上腺素处理后细胞张力降低，随后用激动剂卡巴胆碱处理可克服这些松弛作用。收缩反应不会随着培养时间的延长或重复传代而减弱。收缩蛋白肌球蛋白轻链的磷酸化也作为激动剂诱导收缩的生化指标进行了测量。通过二维凝胶电泳分析，用氯化钾去极化或暴露于卡巴胆碱的细胞显示肌球蛋白磷酸化增加。这些反应在培养7代和9周后仍保持完好。这些结果表明，培养的血管平滑肌细胞在长期培养下不一定会发生表型调节并丧失收缩能力。
