Diglio C A, Grammas P, Giacomelli F, Wiener J
J Cell Physiol. 1986 Nov;129(2):131-41. doi: 10.1002/jcp.1041290202.
This report describes the development and establishment of long-term serial cultures of adult rat vascular smooth muscle cells (SMC) derived from cerebrocortical resistance vessels (small arteries and arterioles). Electron microscopic examination of microvessels isolated off a 150 microns nylon mesh sieve clearly demonstrated the predominance of these vessel types. Initial outgrowth from collagenase-elastase-treated microvessel fragments yielded both endothelium and smooth muscle cells. However, at confluency (2-3 weeks) these cultures consisted of a homogeneous population of broad, polygonal cells that grew in a multilayered "hill and valley" pattern typical of SMC in vitro. For comparative morphological and functional studies, SMC cultures were also initiated from rat thoracic aortas utilizing ring segments as explants. The smooth muscle origin of cultures derived from both resistance vessel (RV) and aorta (RA) was further demonstrated by positive immunofluorescent staining by the specific smooth muscle alpha-actin and myosin antibodies. Ultrastructural examination of these SMC cultures revealed similar morphologic features consisting of typical cytoplasmic myofilament bundles with associated dense bodies and numerous pinocytotic vesicles. Cell growth studies on early (less than P 15)- and late (greater than P 15)-passage RV- and RA-SMC populations revealed markedly different cell growth responses. Representative growth curves of early- and late-passage RA-SMC showed a significantly higher growth rate (two- to fourfold) than RV-SMC cultures. Both cultures, however, exhibited a marked increase in growth potential at higher passage levels. Heparin, at a concentration of 100 micrograms/ml inhibited the growth of RV-SMC during the first 3 days after addition in both exponential and growth-arrested culture states, whereas RA-SMC cultures showed no inhibitory response. These studies indicate that long-term RV-SMC cultures can serve as a useful model system to study functional and metabolic properties of this cell type and provide the means to explore further the heterogeneity of SMC derived from different vasculatures in normal as well as various disease states.
本报告描述了源自大脑皮质阻力血管（小动脉和微动脉）的成年大鼠血管平滑肌细胞（SMC）长期连续培养物的建立与发展。对通过150微米尼龙筛网分离出的微血管进行电子显微镜检查，清楚地显示了这些血管类型的优势。胶原酶-弹性蛋白酶处理过的微血管片段最初长出的细胞既有内皮细胞也有平滑肌细胞。然而，在汇合时（2-3周），这些培养物由均匀的、宽大的多边形细胞群体组成，它们以体外SMC典型的多层“峰谷”模式生长。为了进行比较形态学和功能研究，还利用大鼠胸主动脉的环形片段作为外植体启动了SMC培养。通过特异性平滑肌α-肌动蛋白和肌球蛋白抗体的阳性免疫荧光染色，进一步证实了源自阻力血管（RV）和主动脉（RA）的培养物的平滑肌来源。对这些SMC培养物的超微结构检查揭示了相似的形态学特征，包括典型的细胞质肌丝束以及相关的致密体和大量的胞饮小泡。对早期（小于第15代）和晚期（大于第15代）传代的RV-SMC和RA-SMC群体进行的细胞生长研究显示出明显不同的细胞生长反应。早期和晚期传代的RA-SMC的代表性生长曲线显示其生长速率（两到四倍）明显高于RV-SMC培养物。然而，两种培养物在更高传代水平时均表现出显著的生长潜力增加。浓度为100微克/毫升的肝素在添加后的前3天抑制了指数生长期和生长停滞期培养状态下的RV-SMC生长，而RA-SMC培养物则无抑制反应。这些研究表明，长期的RV-SMC培养物可作为一个有用的模型系统，用于研究这种细胞类型的功能和代谢特性，并为进一步探索正常以及各种疾病状态下源自不同脉管系统的SMC的异质性提供手段。
