牛角膜细胞和角膜成纤维细胞的蛋白聚糖合成：培养中角膜细胞表型的维持。

Proteoglycan synthesis by bovine keratocytes and corneal fibroblasts: maintenance of the keratocyte phenotype in culture.

作者信息

Beales M P, Funderburgh J L, Jester J V, Hassell J R

机构信息

Research Department, Shriners Hospitals for Children, Tampa, Florida 33612, USA.

出版信息

Invest Ophthalmol Vis Sci. 1999 Jul;40(8):1658-63.

PMID:10393032

Abstract

PURPOSE

To determine the effect of serum on morphology, growth, and proteoglycan synthesis by primary cultures of collagenase-isolated bovine keratocytes.

METHODS

Keratocytes were isolated from bovine corneas using sequential collagenase digestion and cultured in Dulbecco's modified Eagle's medium (DMEM), with and without fetal bovine serum (FBS). Proteoglycans synthesized by the cells in culture and by keratocytes in intact cornea culture were metabolically radiolabeled with 35SO4. The proteoglycans were characterized by their sensitivity to keratanase, chondroitinase ABC, and heparatinase and by their size on Superose 6 HR. Cell number was determined by measuring DNA content of the culture dishes.

RESULTS

Keratocytes cultured in 10% FBS proliferated, appeared fibroblastic, and synthesized only 9% of the total glycosaminoglycan as keratan sulfate (KS), whereas cells in serum-free media were quiescent, appeared dendritic, and synthesized 47% KS, a value similar to the 45% KS for corneas radiolabeled overnight in organ culture. This increased proportion of KS synthesis in serum-free media was caused by a moderate increase in KS synthesis combined with a substantial decrease in chondroitin sulfate (CS) synthesis. Fractionation on Superose 6 High Resolution showed the size and relative amounts of the CS- and KS-containing proteoglycans synthesized by keratocytes in serum-free media also more closely resembled that of keratocytes in corneas in organ culture than keratocytes in media containing serum.

CONCLUSIONS

A comparison of proteoglycan synthesis and cell morphology between keratocytes in corneas in organ culture and in cell culture indicates that keratocytes maintain a more native biosynthetic phenotype and appearance when cultured in serum-free media. These results also suggest that culturing in the presence of serum fundamentally alters the keratocyte phenotype to an activated cell, mimicking certain changes observed during wound healing.

摘要

目的

确定血清对经胶原酶分离的牛角膜细胞原代培养物的形态、生长及蛋白聚糖合成的影响。

方法

使用连续胶原酶消化法从牛角膜中分离角膜细胞，并在含和不含胎牛血清（FBS）的杜氏改良 Eagle 培养基（DMEM）中培养。培养细胞及完整角膜培养中的角膜细胞合成的蛋白聚糖用³⁵SO₄进行代谢性放射性标记。通过蛋白聚糖对角蛋白酶、软骨素酶 ABC 和肝素酶的敏感性及其在 Superose 6 HR 上的大小来对其进行表征。通过测量培养皿中的 DNA 含量来确定细胞数量。

结果

在 10% FBS 中培养的角膜细胞增殖，呈成纤维细胞样，仅合成占总糖胺聚糖 9%的硫酸角质素（KS），而无血清培养基中的细胞静止，呈树突状，合成 47%的 KS，该值与器官培养中过夜放射性标记的角膜的 45% KS 相似。无血清培养基中 KS 合成比例的增加是由于 KS 合成适度增加以及硫酸软骨素（CS）合成大幅减少所致。在 Superose 6 高分辨率柱上的分级分离显示，无血清培养基中角膜细胞合成的含 CS 和 KS 的蛋白聚糖的大小和相对含量也比含血清培养基中的角膜细胞更类似于器官培养中角膜的角膜细胞。