Tanswell A K, Han R N, Jassal D, Fraher L J, Post M
Department of Paediatrics, Hospital for Sick Children Research Institute, Toronto, Ontario, Canada.
J Dev Physiol. 1991 Apr;15(4):199-209.
Small vessel pulmonary endothelial cells were obtained from rat fetal lung at day 20 of gestation, and were maintained in culture to passage three for study. Endothelial cells grown on a collagen matrix with Dulbecco's minimal essential medium: Ham's F12 medium (1:1, v/v) supplemented with 20 ml/l fetal bovine serum, bovine pituitary extract (50 mg/l), endothelial cell growth supplement (100 mg/l), hydrocortisone (1 mg/l) and an increased (10 mmol/l) magnesium concentration retained the characteristic endothelial cell marker factor VIII antigen during the third passage in culture. The factors responsible for small vessel growth in the developing fetal lung are unknown. To test the hypothesis that small vessel pulmonary endothelial cells would respond to autocrine or paracrine growth factors the effects of conditioned media from fetal lung endothelial cells, fibroblasts and pneumocytes from lungs of the same gestational age were studied in vitro. None of the tested conditioned media had any effect on endothelial cell DNA synthesis in the presence of 20 ml/l fetal bovine serum. Since no paracrine or autocrine effects of conditioned media were observed, the effect of other growth factors that could be derived from the circulation, or from storage sites in subcellular matrix, were studied for effect. When endothelial cells were studied in the presence of 20 ml/l fetal bovine serum and 100 mg/l endothelial cell growth supplement they had enhanced DNA synthesis in response to the progression-type growth factors insulin (5 mg/l), insulin-like growth factor-I and insulin-like growth factor-II (20 micrograms/l) and epidermal growth factor (10 micrograms/l). In the absence of serum or endothelial growth supplement endothelial cell DNA synthesis was enhanced by the competence-type growth factors acidic and basic fibroblastic growth factors at 100 micrograms/l and platelet derived growth factor at 10 micrograms/l. In the absence of exogenous competence-type growth factors neutralizing antibodies to basic fibroblast growth factor reduce DNA synthesis. Of various cytokines tested only interleukin-1 (1 x 10(3) U/l) and tumor necrosis factor (25 x 10(4) U/l) had an effect on endothelial cell DNA synthesis. Endothelial cell division during fetal lung development may be controlled by progression growth factors present in serum, and by either autocrine release of the competence factor basic fibroblast growth factor or paracrine release of platelet-derived growth factor by other cell types.
在妊娠第20天从大鼠胎肺获取小血管肺内皮细胞，并在培养中维持至第三代用于研究。内皮细胞生长在含有杜尔贝科改良伊格尔培养基：哈姆F12培养基（1:1，v/v）的胶原基质上，该培养基补充有20 ml/l胎牛血清、牛垂体提取物（50 mg/l）、内皮细胞生长补充剂（100 mg/l）、氢化可的松（1 mg/l）以及增加的（10 mmol/l）镁浓度，在培养的第三代期间保留了特征性内皮细胞标志物因子VIII抗原。发育中的胎肺中小血管生长的相关因素尚不清楚。为了检验小血管肺内皮细胞会对自分泌或旁分泌生长因子作出反应这一假设，研究了来自相同胎龄肺的胎肺内皮细胞、成纤维细胞和肺细胞的条件培养基在体外的作用。在存在20 ml/l胎牛血清的情况下，所测试的条件培养基均对内皮细胞DNA合成无任何影响。由于未观察到条件培养基的旁分泌或自分泌作用，因此研究了可能源自循环或亚细胞基质储存部位的其他生长因子的作用。当在内皮细胞存在20 ml/l胎牛血清和100 mg/l内皮细胞生长补充剂的情况下进行研究时，它们对促有丝分裂型生长因子胰岛素（5 mg/l）、胰岛素样生长因子-I和胰岛素样生长因子-II（20微克/l）以及表皮生长因子（10微克/l）有增强的DNA合成反应。在无血清或内皮生长补充剂的情况下，内皮细胞DNA合成通过100微克/l的酸性和碱性成纤维细胞生长因子以及10微克/l的血小板衍生生长因子等促增殖型生长因子而增强。在无外源性促增殖型生长因子的情况下，针对碱性成纤维细胞生长因子的中和抗体可减少DNA合成。在所测试的各种细胞因子中，只有白细胞介素-1（1×10³ U/l）和肿瘤坏死因子（25×10⁴ U/l）对内皮细胞DNA合成有影响。胎肺发育过程中的内皮细胞分裂可能受血清中存在的促有丝分裂生长因子以及促增殖因子碱性成纤维细胞生长因子的自分泌释放或其他细胞类型血小板衍生生长因子的旁分泌释放的控制。
