Corneal endothelial modulation: bFGF as direct mediator and corneal endothelium modulation factor as inducer.
Corneal endothelial modulation: bFGF as direct mediator and corneal endothelium modulation factor as inducer.
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发表时间：
1994-04
影响因子：
4.4
通讯作者：
E. P. Kay;Xin Gu;Ronald E. Smith
中科院分区：
文献类型：
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作者：
E. P. Kay;Xin Gu;Ronald E. Smith
目的本实验室以往报道的角膜内皮调节因子有两种：碱性成纤维细胞生长因子(BFGF)和炎症细胞释放的角膜内皮调节因子(CEMF)。由这两个不同因素介导的表型改变是相同的--细胞增殖显著增加、细胞形状改变和纤维状胶原蛋白的合成。目前的研究试图确定碱性成纤维细胞生长因子是否是角膜内皮调节的直接介质，以及CEMF是否在诱导碱性成纤维细胞生长因子的产生中起作用。方法采用免疫印迹法检测CEMF介导的碱性成纤维细胞生长因子的合成，放线菌酮阻断蛋白质合成。使用碱性成纤维细胞生长因子特异性反义寡核苷酸引物抑制CEMF介导的碱性成纤维细胞生长因子的合成，进一步阻断碱性成纤维细胞生长因子的自分泌活性。细胞计数法检测细胞增殖情况。通过Northern印迹分析确定RNA的稳态水平。通过顺序电泳法、洗脱法和蛋白质复性法进一步纯化了CEMF。结果通过对静止的角膜内皮细胞的生长促进作用来衡量CEMF和bFGF对角膜内皮细胞的协同作用。在任何给定的CEMF浓度下，碱性成纤维细胞生长因子介导的细胞增殖呈剂量依赖性。因此，10 ng/ml的碱性成纤维细胞生长因子和2.5微克/毫升的CEMF显示出饱和的对内皮细胞增殖的协同作用。当在这些条件下测量胶原RNA的稳态水平时，未处理的细胞显示出5.6和5.0kb的α2(I)胶原RNA的双倍体。与对照组相比，碱性成纤维细胞生长因子和CEMF同时处理的细胞表达的转录水平大多较低，而对照组细胞的转录水平较高。然而，6.9kb的IV型胶原RNA水平在定性或定量上没有明显变化，但在碱性成纤维细胞生长因子和CEMF处理的细胞中，α2(IV)胶原RNA的水平最低。外源性碱性成纤维细胞生长因子和CEMF均不能诱导角膜内皮细胞中bFGF信使RNA的表达，而同时用bFGF和CEMF处理则选择性地增加4.9kb转录本的表达。当蛋白质合成被放线菌亚胺抑制时，在CEMF存在的情况下，bFGF的合成被阻断，导致角膜内皮调节的抑制。分析碱性成纤维细胞生长因子反义引物对内皮细胞生长的影响。反义寡核苷酸可阻断50%的反义寡核苷酸对CEMF诱导的碱性成纤维细胞生长因子的增强作用。最后对CEMF进行了纯化，纯化后的蛋白大小约为17kD，具有调节活性。结论CEMF诱导的bFGF从头合成是角膜内皮调节所必需的。
PURPOSE Previously reported from this laboratory are two distinct factors responsible for corneal endothelium modulation: basic fibroblast growth factor (bFGF) and the corneal endothelium modulation factor (CEMF) that is released by inflammatory cells. The altered phenotypes mediated by these two distinct factors--marked increase in cell proliferation, cell shape changes, and synthesis of fibrillar collagens--are identical. The current study sought to determine if bFGF is the direct mediator for corneal endothelium modulation and if CEMF plays a role in inducing bFGF production. METHODS bFGF synthesis mediated by CEMF was analyzed by immunoblot assay; cycloheximide was used to block protein synthesis. bFGF-Specific antisense oligonucleotide primer was used to inhibit CEMF-mediated bFGF synthesis and to block further the autocrine activity of bFGF. Cell proliferation was measured by cell counting. The steady-state levels of RNA were determined by Northern blot analysis. CEMF was further purified to homogeneity by sequential electrophoresis, elution, and renaturation of protein. RESULTS The synergistic effect of CEMF and bFGF on corneal endothelial cells was measured by their growth-promoting activity on quiescent corneal endothelial cells. There was a dose-dependent cell proliferation mediated by bFGF at any given CEMF concentration. Thus, bFGF at 10 ng/ml with CEMF at 2.5 micrograms/ml demonstrated saturable synergistic activity on endothelial cell proliferation. When the steady-state levels of collagen RNA were measured under these conditions, the untreated cells showed the doublets of 5.6 and 5.0 kb of alpha 2(I) collagen RNA. The cells treated simultaneously with bFGF and CEMF contained mostly lower transcript, compared to the significant level of upper transcript in control cells. However, there was no significant change in the level of 6.9-kb type IV collagen RNA qualitatively or quantitatively; nonetheless, the level of alpha 2(IV) collagen RNA was lowest in cells treated with bFGF plus CEMF. Neither exogenous bFGF nor CEMF caused induction of bFGF messenger RNA in corneal endothelial cells, whereas simultaneous treatment with bFGF and CEMF selectively enhanced the 4.9-kb transcript. When protein synthesis was inhibited by cycloheximide, bFGF synthesis was blocked in the presence of CEMF, leading to inhibition of corneal endothelium modulation. The effect on endothelial cell growth of bFGF antisense primer was analyzed. Antisense primer blocked by 50% the enhanced growth potential mediated by bFGF induced with CEMF. Finally, CEMF was purified to homogeneity: the purified protein is approximately 17 kD and assumes the modulating activities. CONCLUSIONS These findings suggest that de novo synthesis of bFGF induced by CEMF is required for corneal endothelium modulation.