Proliferative response of corneal endothelial cells from young and older donors
Proliferative response of corneal endothelial cells from young and older donors
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DOI：
10.1167/iovs.03-0814
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发表时间：
2004-06-01
影响因子：
4.4
通讯作者：
Joyce, NC
中科院分区：
文献类型：
--
作者：
Zhu, C;Joyce, NC
目的.比较表皮生长因子（EGF）、神经生长因子（NGF）、血小板源性生长因子-BB（PDGF-BB）、牛垂体提取物和胎牛血清（FBS）单独或联合应用对年轻人（50岁）角膜内皮细胞（HCEC）增殖的影响。来自2至79岁供体的角膜获自国家疾病研究交流中心。解剖具有完整内皮的后弹力膜。通过EDTA处理分离细胞并培养至汇合。HCEC标志物抗体9.3.E检测纯内皮细胞群。在培养的细胞达到汇合之前或之后测试抗体Ki 67和ZO-1以指示细胞增殖和细胞-细胞接触形成。倒置相差显微镜观察细胞形态。传代I至VII用于测试各种因素对细胞增殖的影响。对于每项研究，接种相同数量的细胞，在4% FBS中保持过夜以允许细胞附着，洗涤，并在以下之一中孵育长达3周：单独的改良Eagle最小必需培养基（Opti-MEM-I）; Opti-MEM-I加EGF、NGF、PDGF-BB、牛垂体提取物或FBS;或因子组合。在接种后的不同时间，通过电子细胞计数器测定细胞数。对于每种条件，检测三个单独的威尔斯孔，并对每个样品计数三次。使用来自不同供体和年龄组的细胞重复研究至少两次。在每项研究中，进行单因素ANOVA检验以分析统计学显著性。细胞用抗体9.3.E染色呈阳性，表明HCEC的分离和缺乏上皮细胞或角膜细胞的污染。在亚融合培养物中发现Ki 67阳性染色，表明细胞处于周期性变化。质膜相关ZO-1染色和Ki 67染色的缺乏表明培养的细胞形成接触抑制单层。培养的细胞密度降低，大小增加，并变得更加异质性取决于供体年龄和传代次数。在OptiMEM-I中孵育促进附着并诱导高于MEM的中度增殖反应（P < 0.001）。一般来说，对生长刺激的增殖反应相对缓慢，细胞计数通常在暴露于生长促进剂后10至14天达到平台。EGF产生广泛的剂量依赖性效应，并且在5-50 ng/mL时，峰值细胞计数显著高于基础水平（P < 0.001）。EGF持续刺激来自年轻供体的细胞增殖，但在刺激来自老年供体的细胞生长方面效果较差。在任何测试浓度下，NGF均未显示出一致的显著刺激作用。在来自同一供体的培养物中，PDGF-BB（25 ng/mL）倾向于比EGF更大程度地刺激生长（P < 0.05）。在1.0（P < 0.05）至100 μ g/mL（P < 0.001）时，补骨脂提取物显著增加计数。PDGF-BB与垂体提取物联合应用的刺激作用明显强于垂体提取物（P < 0.01）或单独应用PDGF-BB（P < 0.01）。FBS（1%-8%）以剂量依赖性方式增加细胞数量，并且在4%-8%时，产生的计数显著高于任何单一生长促进剂。来自年轻和老年供体的HCEC可以在体外增殖以响应生长促进剂。当达到汇合时，在多种有丝分裂原存在下的增殖停止，表明形成了接触抑制单层。一般来说，从年轻供体培养的细胞对测试的试剂更敏感，但HCEC对这些试剂的相对反应相似，与供体年龄无关。相同细胞群对不同有丝分裂原的反应程度的相对差异表明，这些有丝分裂原诱导不同的下游信号。HCEC对FBS的相对稳健的增殖反应可能涉及刺激多个下游信号传导途径和/或诱导比测试的其他生长促进剂更持续的下游信号传导。
PURPOSE. To compare the effect of epidermal growth factor (EGF), nerve growth factor (NGF), platelet-derived growth factor-BB (PDGF-BB), bovine pituitary extract, and fetal bovine serum (FBS), alone or in combination, on proliferation of human corneal endothelial cells (HCEC) cultured from young (50 years old).METHODS. Corneas from donors 2 to 79 years old were obtained from the National Disease Research Interchange. Descemet's membrane with intact endothelium was dissected. Cells were isolated by EDTA treatment and cultured to confluence. The HCEC marker, antibody 9.3.E, tested for pure endothelial populations. Antibody Ki67 and ZO-1 tested either before or after cultured cells reached confluence to indicate cell proliferation and cell-cell contact formation. Cell morphology was documented by inverted phase-contrast microscopy. Passages I through VII were used to test the effect of various factors on cell proliferation. For each study, equal numbers of cells were seeded, maintained overnight in 4% FBS to permit cell attachment, washed, and incubated for up to 3 weeks in one of the following: modified Eagle's Minimum Essential Medium (Opti-MEM-I) alone; Opti-MEM-I plus EGF, NGF, PDGF-BB, bovine pituitary extract, or FBS; or a combination of factors. At various times after seeding, cell numbers were determined by electronic cell counter. For each condition, three separate wells were tested and each sample was counted three times. Studies were repeated at least twice using cells from different donors and age groups. Within each study, a one-way ANOVA test was performed to analyze statistical significance.RESULTS. Cells stained positively with antibody 9.3.E, indicating isolation of HCEC and lack of contamination with epithelial cells or keratocytes. Positive staining of Ki67, indicating cycling cells, was found in subconfluent cultures. Plasma membrane-associated ZO-1 staining and lack of Ki67 staining indicated that cultured cells formed a contact-inhibited monolayer. Cultured cells decreased in density, increased in size, and became more heterogeneous depending on donor age and on the number of passages. Incubation in OptiMEM-I promoted attachment and induced a moderate proliferative response above that of MEM (P < 0.001). In general, proliferative responses to growth stimuli were relatively slow, with cell counts generally plateauing 10 to 14 days after exposure to growth-promoting agents. EGF yielded a broad, dose-dependent effect and, at 5-50 ng/mL, peak cell counts were significantly higher (P < 0.001) than basal levels. EGF consistently stimulated proliferation in cells from younger donors, but was less effective in stimulating growth of cells from older donors. NGF did not show a consistent significant stimulatory effect at any concentration tested. PDGF-BB (25 ng/mL) tended to stimulate growth to a greater extent than EGF (P < 0.05) in cultures from the same donor. Pituitary extract significantly increased counts at 1.0 (P < 0.05) to 100 ug/mL (P < 0.001). PDGF-BB plus pituitary extract demonstrated greater stimulation than pituitary extract (P < 0.01) or PDGF-BB alone (P < 0.01). FBS (1%-8%) increased cell numbers in a dose-dependent manner, and, at 4%-8%, yielded counts significantly higher (P < 0.001) than that of any single growth-promoting agent tested.CONCLUSIONS. HCEC from both young and older donors can proliferate in vitro in response to growth-promoting agents. Proliferation in the presence of multiple mitogens ceased when confluence was reached, indicating the formation of a contact-inhibited monolayer. In general, cells cultured from young donors were more responsive to the agents tested, but the relative response of HCEC to these agents was similar, regardless of donor age. The relative difference in the extent of the response of the same cell population to different mitogens suggests that these mitogens induce different downstream signals. The relatively robust proliferative response of HCEC to FBS may involve stimulation of multiple downstream signaling pathways and/or induce more sustained downstream signaling than the other growth-promoting agents tested.