STIMULATION OF CORNEAL ENDOTHELIAL CELL-PROLIFERATION INVITRO BY FIBROBLAST AND EPIDERMAL GROWTH-FACTORS
STIMULATION OF CORNEAL ENDOTHELIAL CELL-PROLIFERATION INVITRO BY FIBROBLAST AND EPIDERMAL GROWTH-FACTORS
复制标题
DOI：
10.1016/0014-4835(77)90248-2
复制
发表时间：
1977-01-01
影响因子：
3.4
通讯作者：
BIRDWELL, CR
中科院分区：
文献类型：
--
作者：
GOSPODAROWICZ, D;MESCHER, AL;BIRDWELL, CR
建立了牛角膜内皮细胞培养体系，以研究角膜内皮细胞增殖的调控因素。通过用带凹槽的导向器轻轻刮擦角膜后表面来制备细胞。然后将内皮节段悬浮在含有10%小牛血清和100 ng/ml成纤维细胞生长因子（FGF）的DME中。4天后，角膜内皮细胞附着在组织培养皿上，产生快速分裂的小细胞集落。用AgNO 3染色的固定培养物acentuise细胞间功能揭示了单层内多边形细胞的紧密贴壁性质，并且卵圆形核偏心地定位在每个细胞中。这种形态与体内角膜内皮观察到的形态相似。体外培养的牛角膜内皮细胞的超微结构是典型的高代谢细胞。细胞内有大量的线粒体、多聚核糖体和微丝束。超微结构和阿辛绿色组织化学染色的培养物表明，细胞在培养中产生细胞外的胶原物质。成纤维细胞生长因子（FGF）和表皮生长因子（EGF）在体外对牛角膜内皮细胞有促有丝分裂作用。在5%牛血浆存在下维持在低密度的细胞分裂，倍增时间为48 h;加入EGF或FGF将细胞倍增时间降低至20-24 h。在任一种生长因子存在下达到的最终细胞密度为10 × 104。仅在5%血浆中维持的细胞。在10%小牛血清存在下，角膜内皮细胞达到与在1%血清加FGF中获得的密度相同的最终密度，并且向10%血清中加入FGF导致最终密度为3.5倍。高于单独用10%血清观察到的。将FGF包含在含有血清的DME培养基中允许从以低密度（0.6个细胞/cm 2）接种的培养物中克隆角膜内皮细胞。用EGF刺激增殖的半最大反应为1.5 × 10 - 6。10- 10 M和6 × 10 - 10 M的FGF。10- 10米。EGF和FGF刺激静息角膜内皮细胞中的DNA合成，EGF从1.5 × 10 - 6开始有效。10-15至1.5 ×10-13和FGF从7 × 10 - 13起有效。10-12至7倍10-9 M.放射自显影表明，FGF和EGF，像血清，刺激细胞群体作为一个整体，启动DNA合成。
Bovine corneal endothelial cell culture was developed to study the factors controlling corneal endothelium proliferation. Cells were prepared by scraping the posterior corneal surface gently with a grooved director. Endothelial segments were then suspended in DME with 10% calf serum and 100 ng/ml of fibroblast growth factor (FGF). After 4 days, corneal endothelial cells attached to the tissue culture dish, giving rise to colonies of small, rapidly dividing cells. Fixed cultures stained with AgNO3 to acentuate the intercellular function revealed the closely apposed nature of the polygonal cells within the monolayer, and the ovoid nucleus positioned eccentrically in each cell. This morphology was similar to that observed with the corneal endothelium in vivo. Ultrastructure of bovine corneal endothelial cells grown in vitro was typical of cells with high metabolic rates. Cells had numerous mitochondria, polysomes and microfilament bundles. Ultrastructure and histochemical staining of the culture with alcian green demonstrated that cells maintained in culture produced extracellular collagenous material. Fibroblast growth factor (FGF) and epidermal growth factor (EGF) were mitogenic in vitro for endothelial cells derived from bovine corneas. Cells maintained at low density in the presence of 5% bovine plasma divided with a doubling time of 48 h; addition of EGF or FGF reduced cell doubling time to 20-24 h. Final cell density reached in the presence of either growth factor was 10 .times. that of cells maintained in 5% plasma alone. In the presence of 10% calf serum, corneal endothelial cells reached a final density identical to that obtained in 1% serum plus FGF, and addition of FGF to 10% serum resulted in a final density 3.times. higher than that observed with 10% serum alone. Inclusion of FGF in DME medium containing serum permitted cloning of corneal endothelial cells from cultures seeded at low density (0.6 cells/cm2). Half-maximal response for stimulation of proliferation with EGF was at 1.5 .times. 10-10M and with FGF at 6 .times. 10-10M. EGF and FGF stimulated DNA synthesis in resting corneal endothelial cells, with EGF effective from 1.5 .times. 10-15 to 1.5 .times. 10-13 and FGF effective from 7 .times. 10-12 to 7 .times. 10-9 M. Autoradiography demonstrated that FGF and EGF, like serum, stimulated the cell population as a whole to initiate DNA synthesis.