Maskin S L, Tseng S C
Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami School of Medicine, Florida.
Invest Ophthalmol Vis Sci. 1992 Jan;33(1):205-17.
We have established a serum-free clonal culture system to study the growth and differentiation of individual progenitor epithelial cells of the meibomian gland independent of other cell types and undefined serum factors. Single meibomian gland epithelial cells were obtained by subjecting whole meibomian glands to a brief EDTA treatment, needle aspiration, and nylon mesh filtration. The cells had been isolated by a previously described method using enzymatic and microsurgical techniques. Four to five hundred cells obtained were seeded on a 35 mm or 60 mm dish with serum-free MCDB 151 medium containing insulin, transferrin, selenium, ethanolamine, o-phosphorylethanolamine, dimethyl sulfoxide, and calcium. Control cultures with this basic medium did not show continuous clonal growth. Addition of epidermal growth factor (EGF) from 1 to 5 and 10 ng/ml enhanced clonal growth with a decreasing effect as measured by colony forming efficacy and colony size. Clonal growth was associated with the occurrence of two types of colony morphology and an increase in intracellular lipid production in the BrdU-labelled cells, shown by Nile red fluorescent staining. In contrast, the clonal growth stimulated by addition of acidic fibroblast growth factor (aFGF) from 1 to 100 ng/ml exhibited a pattern of an initial increase followed by a decrease, with the maximum noted at 10 ng/ml. Moreover, the aFGF-stimulated clonal growth was associated with a uniform colony morphology and minimal lipid synthesis even in the non-BrdU-labelled cells. These results indicate that clonal growth of meibomian gland epithelium can be achieved in a serum-free culture by adding either of these two peptide growth factors. Furthermore, the clonal growth stimulated by EGF was associated with progressive cellular differentiation more so than that of aFGF. Further exploration of such a differential regulation of proliferation and differentiation by EGF and aFGF may provide a better understanding of normal meibomian gland function and pathogenesis of various meibomian gland disorders.
我们建立了一种无血清克隆培养系统，以研究睑板腺单个祖上皮细胞的生长和分化，该系统独立于其他细胞类型和未定义的血清因子。通过对整个睑板腺进行短暂的乙二胺四乙酸（EDTA）处理、针吸和尼龙网过滤，获得单个睑板腺上皮细胞。这些细胞是通过先前描述的使用酶促和显微手术技术的方法分离得到的。将获得的400至500个细胞接种在含有胰岛素、转铁蛋白、硒、乙醇胺、邻磷酸乙醇胺、二甲基亚砜和钙的无血清MCDB 151培养基的35毫米或60毫米培养皿上。使用这种基础培养基的对照培养物未显示出连续的克隆生长。添加1至5和10纳克/毫升的表皮生长因子（EGF）可增强克隆生长，通过集落形成效率和集落大小测量，其效果呈递减趋势。克隆生长与两种集落形态的出现以及经BrdU标记的细胞内脂质产生的增加相关，尼罗红荧光染色显示了这一点。相比之下，添加1至100纳克/毫升的酸性成纤维细胞生长因子（aFGF）刺激的克隆生长呈现出先增加后减少的模式，在10纳克/毫升时达到最大值。此外，aFGF刺激的克隆生长与均匀的集落形态和即使在未用BrdU标记的细胞中也极少的脂质合成相关。这些结果表明，通过添加这两种肽生长因子中的任何一种，均可在无血清培养中实现睑板腺上皮的克隆生长。此外，与aFGF相比，EGF刺激的克隆生长与细胞的渐进性分化关联更大。进一步探索EGF和aFGF对增殖和分化的这种差异调节，可能有助于更好地理解睑板腺的正常功能以及各种睑板腺疾病的发病机制。
