Diglio C A, Grammas P, Giacomelli F, Wiener J
Department of Pathology, Wayne State University, School of Medicine, Detroit, Michigan 48201.
Tissue Cell. 1988;20(4):477-92. doi: 10.1016/0040-8166(88)90051-1.
This report describes the initiation, cloning and establishment of long-term serial cultures of rat heart-derived vascular endothelial (EC) and smooth muscle cells (SMC). Populations of these cells derived from both the macro-and microcirculation were obtained utilizing isolated heart perfusion technique. Elimination of potential mesothelial cell contamination was achieved by ethanol fixation of the pericardial surface prior to perfusion. Initial outgrowths from perfusate yielded both endothelial (rapid adhering) and smooth muscle (slow adhering) appearing cell populations. Subsequent pooling of individual EC colonies resulted in maintaining, with gradual subcultivation, a stable homogeneous population which was designated RHE-parent. Upon continual subculture late passage (greater than P10) RHE-parent cell cultures expressed a marked heterogeneity in endothelial phenotypes. Cloning experiments resulted in establishing two distinct EC populations designated RHE-clone 1A and RHE-clone 2A. All RHE cell cultures exhibited the typical cobblestone growth pattern and positive immunofluorescent staining for factor VIII related antigen. In contrast, rat heart-derived smooth muscle cell (RH-SMC) cultures displayed the typical multilayered 'hill and valley' pattern and positive fluorescence for SMC-specific actin and myosin antibodies. Additional EC preparations, obtained without prior fixation of the pericardial surface, revealed cell clusters which stained positive for cytokeratin. On the other hand, RHE parent and cloned populations stained exclusively for vimentin, further confirming the absence of mesothelial cell contamination in these cultures. Cell growth studies on early (less than P10) and late (greater than P10) passage RHE-parent population revealed markedly different cell growth responses and cell morphology. Both EC cloned populations and more notably RHE-parent (less than P10) cultures were capable of significant growth when maintained in limiting serum concentration. Growth studies using serum-free RHE-parent conditioned medium demonstrated mitogenic activity when tested on RHE-parent cultures indicating the presence of an endothelial cell-derived growth factor. These studies indicate that long-term RHE and RH-SMC derived cell cultures can serve as a useful model to study the biology of vascular cells derived from different sites. In addition the demonstration of mitogenic activity in these cultures will enable us to explore further the nature of this response and compare this phenomenon with growth factors identified in large vessel cell systems.
本报告描述了大鼠心脏来源的血管内皮细胞（EC）和平滑肌细胞（SMC）长期连续培养物的起始、克隆及建立过程。利用离体心脏灌注技术获得了源自大循环和微循环的这些细胞群体。在灌注前通过乙醇固定心包表面，消除了潜在的间皮细胞污染。灌注液最初长出的细胞既有呈内皮细胞样（快速黏附）的群体，也有呈平滑肌细胞样（缓慢黏附）的群体。随后将单个EC集落合并，经逐步传代培养，维持了一个稳定的均一群体，命名为RHE-亲代。在连续传代至晚期传代（大于第10代）时，RHE-亲代细胞培养物在内皮细胞表型上表现出明显的异质性。克隆实验建立了两个不同的EC群体，命名为RHE-克隆1A和RHE-克隆2A。所有RHE细胞培养物均呈现典型的鹅卵石样生长模式，且对VIII因子相关抗原呈阳性免疫荧光染色。相比之下，大鼠心脏来源的平滑肌细胞（RH-SMC）培养物呈现典型的多层“峰谷”模式，且对SMC特异性肌动蛋白和肌球蛋白抗体呈阳性荧光。在未事先固定心包表面的情况下获得的其他EC制剂显示，细胞簇对角蛋白呈阳性染色。另一方面，RHE亲代群体和克隆群体仅对波形蛋白呈阳性染色，进一步证实这些培养物中不存在间皮细胞污染。对早期传代（小于第10代）和晚期传代（大于第10代）的RHE-亲代群体进行的细胞生长研究显示，细胞生长反应和细胞形态存在显著差异。当维持在低血清浓度时，EC克隆群体尤其是RHE-亲代（小于第10代）培养物均能显著生长。使用无血清RHE-亲代条件培养基进行的生长研究表明，在RHE-亲代培养物上进行测试时，其具有促有丝分裂活性，表明存在一种内皮细胞来源的生长因子。这些研究表明，长期的RHE和RH-SMC来源的细胞培养物可作为研究源自不同部位的血管细胞生物学的有用模型。此外，这些培养物中促有丝分裂活性的证明将使我们能够进一步探索这种反应的本质，并将这一现象与在大血管细胞系统中鉴定出的生长因子进行比较。
