Crisp T M, Alexander J S
Prog Clin Biol Res. 1981;59B:49-70.
The purpose of this study was to define optimal substrate conditions for permitting correlation of cell surface morphology with progesterone secretion by luteinized granulosa cells in vitro. Some 3-5 x 10(5) granulosa cells were obtained from preovulatory follicles of PMS-LH treated rats and plated upon carbon, poly-L-lysine or collagen-coated glass coverslips within plastic multiwells or upon Cytodex, Biosilon or poly-L-lysine coated Sephadex beads within siliconized glass roller tubes. Cells were maintained at 36.5 degrees C in 1.0 ml medium with and without 1.0 microgram purified prolactin (r, bPRL). Spent medium was assayed for progesterone by RIA. Cultures were processed for SEM observation of cell surface features. Results indicate that: 1) cells cultured in multiwells upon carbon, poly-L-lysine or glass grow as flattened monolayers but demonstrate considerable tearing distortion following routine processing for SEM, 2) cells cultured upon collagen-coated coverslips or microcarriers within roller tubes maintain a more spherical shape, exhibit the least distortion of surface fine structure and secrete increased amounts of progesterone in the presence of PRL. It is concluded that the use of Cytodex microcarriers for culturing granulosa cells permits optimal conditions for both progesterone secretion and maintenance of surface morphology.
本研究的目的是确定最佳底物条件，以便在体外使黄体化颗粒细胞的细胞表面形态与孕酮分泌相关联。从经PMS-LH处理的大鼠的排卵前卵泡中获取约3 - 5×10⁵个颗粒细胞，并接种于塑料多孔板内涂有碳、聚-L-赖氨酸或胶原蛋白的玻璃盖玻片上，或接种于硅化玻璃滚筒管内涂有Cytodex、Biosilon或聚-L-赖氨酸的葡聚糖凝胶珠上。细胞在含有和不含有1.0微克纯化催乳素（r，bPRL）的1.0毫升培养基中于36.5℃下培养。用放射免疫分析法测定用过的培养基中的孕酮。对培养物进行处理以通过扫描电子显微镜观察细胞表面特征。结果表明：1）在多孔板中于碳、聚-L-赖氨酸或玻璃上培养的细胞生长为扁平单层，但在常规扫描电子显微镜处理后显示出相当大的撕裂变形；2）在滚筒管内涂有胶原蛋白的盖玻片或微载体上培养的细胞保持更球形的形状，表面精细结构的变形最小，并且在存在催乳素的情况下分泌更多量的孕酮。得出的结论是，使用Cytodex微载体培养颗粒细胞可为孕酮分泌和表面形态的维持提供最佳条件。
