Chen S F, Fei X, Li S H
Department of Pathophysiology, Second Military Medical University, Shanghai, People's Republic of China.
Microvasc Res. 1995 Jul;50(1):119-28. doi: 10.1006/mvre.1995.1044.
Our study indicates that when small pieces of lung or muscles of chest wall are cultured, erythrocytes and leukocytes (PMNs) leave the tissues first, followed by vascular endothelial cells (ECs). Fibroblasts and other mixed cells grow after 72 hr culture. The ECs can then be isolated avoiding mechanical and chemical injuries. The lung tissue is obtained from the peripheral surface and muscles from the chest. It is then cut into pieces and cultured with DMEM containing 20% fetal bovine serum. After 60 hr culture, the tissues are discarded. The flask contains only ECs and blood cells. Blood cells can be cleared out after the cells are subcultured once or twice. The primary cells and the subcultured cells cultured on gelatinized culture dish give the capillary-like structure. Cells cultured on untreated dishes have regular cobblestone morphology and junctional contacts. The isolated cells were not mesothelial cells because the cells did not react to antibody against cytokeratin 18, while mesothelial cells reacted strongly to the antibody. The cells can be isolated from the lung tissue without pleura. The primary microvascular ECs are also cultured on microcarriers (cytodex 3). Because both mechanical and proteolytic injuries are avoided, the cells may be more similar to cells in the in vivo state. There are no significant differences in PMN-endothelium adherence and monolayer responses to second messengers, platelet activating factor, and phospholipase A2 when pulmonary and muscular microvascular endothelial cells are compared.
我们的研究表明，当培养小块肺组织或胸壁肌肉时，红细胞和白细胞（多形核白细胞）首先从组织中逸出，随后是血管内皮细胞。成纤维细胞和其他混合细胞在培养72小时后生长。然后可以分离出内皮细胞，避免机械和化学损伤。肺组织取自外周表面，肌肉取自胸部。然后将其切成小块，用含有20%胎牛血清的DMEM培养基培养。培养60小时后，丢弃组织。培养瓶中仅含有内皮细胞和血细胞。细胞传代培养一两次后，血细胞即可清除。在明胶化培养皿上培养的原代细胞和传代细胞形成毛细血管样结构。在未处理的培养皿上培养的细胞具有规则的鹅卵石形态和连接接触。分离出的细胞不是间皮细胞，因为这些细胞对细胞角蛋白18抗体无反应，而间皮细胞对该抗体反应强烈。这些细胞可以从无胸膜的肺组织中分离出来。原代微血管内皮细胞也在微载体（细胞微载体3）上培养。由于避免了机械和蛋白水解损伤，这些细胞可能更类似于体内状态的细胞。比较肺和肌肉微血管内皮细胞时，多形核白细胞与内皮细胞的黏附以及单层细胞对第二信使、血小板活化因子和磷脂酶A2的反应没有显著差异。
