TGF-beta2 in aqueous humor suppresses S-phase entry in cultured corneal endothelial cells.
TGF-beta2 in aqueous humor suppresses S-phase entry in cultured corneal endothelial cells.
复制标题
DOI：
--
复制
发表时间：
1999-10
影响因子：
4.4
通讯作者：
Ko-Hua Chen;Ko-Hua Chen;D. Harris;N. Joyce
中科院分区：
文献类型：
--
作者：
Ko-Hua Chen;Ko-Hua Chen;D. Harris;N. Joyce
目的体内角膜内皮细胞被阻滞在G1期，这是细胞周期的一个阶段，为细胞的DNA合成做好准备。在许多细胞类型中，转化因子（TGF）-β通过诱导G1期阻滞来抑制增殖。有证据表明，角膜内皮细胞合成TGF-β 1的mRNA，并且也沐浴在含有TGF-β 2（主要以潜伏形式）的房水中。因此，这种细胞因子可以在体内将角膜内皮维持在G1期阻滞状态。这些研究的目的是确定外源性TGF-β 2和房水中TGF-β 2对培养的角膜内皮细胞DNA合成的影响。方法体外培养大鼠角膜内皮细胞，通过形态学和逆转录-聚合酶链反应（RT-PCR）鉴定。通过血清饥饿24小时使亚融合细胞同步于G 0期（静止）。然后在存在或不存在外源性TGF-β 2或活化的大鼠房水的情况下将血清加入到细胞中。加入[3 H]胸苷，并在不同时间点测量放射性以检测DNA合成。将外源性TGF-β 2或活化的大鼠房水与中和抗体预孵育用于测试细胞因子特异性。结果：加入血清后约16小时，[3 H]胸苷掺入量开始线性增加，约24小时出现峰值掺入。将细胞暴露于血清加TGF-β 2以剂量依赖性方式抑制[3 H]胸苷掺入，浓度范围为5 pg/ml至5 ng/ml。[3 H]胸苷掺入在暴露于血清加1：10稀释的大鼠房水的细胞中也受到抑制。中和抗体逆转了外源性TGF-β 2和房水的作用。结论外源性TGF-β 2和房水中TGF-β 2抑制大鼠角膜内皮细胞S期进入。这些结果表明，这种细胞因子在房水中可以帮助维持角膜内皮细胞在G1期阻滞状态在体内。
PURPOSE Corneal endothelium in vivo is arrested in G1, the phase of the cell cycle that prepares cells for DNA synthesis. In many cell types, transforming factor (TGF)-beta inhibits proliferation by inducing G1-phase arrest. Evidence indicates that corneal endothelial cells synthesize mRNA for TGF-beta1 and are also bathed in aqueous humor that contains TGF-beta2 (mainly in a latent form). As such, this cytokine may maintain the corneal endothelium in a G1-phase-arrested state in vivo. The purpose of these studies was to determine the effect of exogenous TGF-beta2 and TGF-beta2 in aqueous humor on DNA synthesis in cultured corneal endothelial cells. METHODS Rat corneal endothelial cells were grown in explant culture and identified by morphology and reverse transcription-polymerase chain reaction using primers for specific corneal cell markers. Subconfluent cells were synchronized in the G0 phase (quiescence) by serum starvation for 24 hours. Serum was then added to the cells in the presence or absence of exogenous TGF-beta2 or activated rat aqueous humor. [3H]Thymidine was added, and radioactivity was measured at various time points to detect DNA synthesis. Preincubation of exogenous TGF-beta2 or activated rat aqueous humor with neutralizing antibody was used to test for cytokine specificity. RESULTS A linear increase in [3H]thymidine incorporation began approximately 16 hours after serum addition, and peak incorporation occurred at approximately 24 hours. Exposure of cells to serum plus TGF-beta2 suppressed [3H]thymidine incorporation in a dose-dependent manner at concentrations ranging from 5 pg/ml to 5 ng/ml. [3H]Thymidine incorporation was also suppressed in cells exposed to serum plus rat aqueous humor diluted 1:10. Neutralizing antibody reversed the effects of both exogenous TGF-beta2 and aqueous humor. CONCLUSIONS Exogenous TGF-beta2 and TGF-beta2 in aqueous humor suppress S-phase entry of rat corneal endothelial cells. These results suggest that this cytokine in aqueous humor could help maintain the corneal endothelium in a G1-phase-arrested state in vivo.