Keithahn M A, Aotaki-Keen A E, Schneeberger S A, Hjelmeland L M, Morse L S
Department of Ophthalmology, University of California, Davis, Sacramento 95816, USA.
Invest Ophthalmol Vis Sci. 1997 Sep;38(10):2073-80.
To demonstrate the expression of fibroblast growth factor-5 (FGF-5) by bovine choroidal microvascular endothelial (BCME) cells and to investigate its possible role as an autocrine mitogen in these cells.
Expression of FGF-5 by BCME cells was studied by a combination of Northern and Western blot analyses. Total RNA was isolated from BCME cultures at passages 5 through 8 and analyzed by Northern blot analysis for the presence of FGF-5 transcripts, using a 1-kb human complemetary DNA. Slot-blot analysis was performed to determine possible cross-reactivity between this probe and acidic and basic FGFs of human and bovine species. A previously characterized antibody directed against the aminoterminus of the human FGF-5 sequence was used in Western blot analyses to identify immunoreactive proteins released by BCME cells into the medium. Finally, the mitogenic activity of human recombinant FGF-5 on a variety of cell types was evaluated, using a cellular proliferation assay.
Northern blot analysis provided evidence for the expression of two major FGF-5 transcripts at 4 kb and 3 kb and two minor transcripts at 2.2 kb and 1.7 kb. A single immunoreactive protein with a molecular weight of 34 kDa was identified by Western blot analysis of conditioned media. In cellular proliferation assays, human recombinant FGF-5 was not mitogenic in BCME cells but exhibited an approximate ED50 of 1.8 to 3.7 nM in BALB/c3T3 fibroblasts. This ED50 was within the range reported by the manufacturer, using a thymidine incorporation assay and a similar embryonic fibroblast cell line. Fibroblast growth factor-5 also stimulated proliferation of human retinal pigment epithelial cells.
Bovine choroidal microvascular endothelial cells exhibit expression in vitro of FGF-5 at the messenger RNA and protein levels. Perivascular and endothelial cell staining for FGF-5 seen previously in choroidal neovascular membranes may therefore arise from expression by choroidal endothelial cells. Because nonglycosylated recombinant FGF-5 does not appear to be a mitogen in BCME cells in vitro, it is reasonable to question its role as an autocrine mitogen in vivo. Fibroblast growth factor-5 may instead be serving paracrine roles in the stimulation of fibroblasts and retinal pigment epithelial cells during the formation of choroidal neovascular membranes. Studies with fully glycosylated recombinant FGF-5 will be required, however, to assess the biologic activity of this member of the FGF gene family.
证实牛脉络膜微血管内皮（BCME）细胞中存在成纤维细胞生长因子-5（FGF-5）的表达，并研究其作为这些细胞自分泌促有丝分裂原的可能作用。
采用Northern印迹和Western印迹分析相结合的方法研究BCME细胞中FGF-5的表达。从第5至8代BCME培养物中分离总RNA，并用1 kb的人互补DNA通过Northern印迹分析检测FGF-5转录本的存在。进行狭缝印迹分析以确定该探针与人及牛的酸性和碱性FGF之间可能的交叉反应性。在Western印迹分析中使用先前鉴定的针对人FGF-5序列氨基末端的抗体，以鉴定BCME细胞释放到培养基中的免疫反应性蛋白。最后，使用细胞增殖测定法评估人重组FGF-5对多种细胞类型的促有丝分裂活性。
Northern印迹分析为4 kb和3 kb处两种主要FGF-5转录本以及2.2 kb和1.7 kb处两种次要转录本的表达提供了证据。通过对条件培养基的Western印迹分析鉴定出一种分子量为34 kDa的单一免疫反应性蛋白。在细胞增殖测定中，人重组FGF-5在BCME细胞中无促有丝分裂作用，但在BALB/c3T3成纤维细胞中表现出约1.8至3.7 nM的半数有效剂量（ED50）。该ED50在制造商使用胸苷掺入测定法和类似的胚胎成纤维细胞系报告的范围内。成纤维细胞生长因子-5也刺激人视网膜色素上皮细胞的增殖。
牛脉络膜微血管内皮细胞在体外的信使RNA和蛋白质水平上均表现出FGF-5的表达。因此，先前在脉络膜新生血管膜中观察到的FGF-5的血管周围和内皮细胞染色可能源于脉络膜内皮细胞的表达。由于非糖基化的重组FGF-5在体外BCME细胞中似乎不是促有丝分裂原，因此有理由质疑其在体内作为自分泌促有丝分裂原的作用。相反，成纤维细胞生长因子-5可能在脉络膜新生血管膜形成过程中对成纤维细胞和视网膜色素上皮细胞的刺激中发挥旁分泌作用。然而，需要对完全糖基化的重组FGF-5进行研究，以评估FGF基因家族这一成员的生物学活性。
