Corcia A, Steinmetz R, Liu J W, Ben-Jonathan N
Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis 46202.
Endocrinology. 1993 Jan;132(1):80-5. doi: 10.1210/endo.132.1.8419149.
Recent evidence suggests that the posterior pituitary (PP), also called the neurointermediate lobe, regulates PRL release. We previously reported that cocultures of anterior pituitary and PP cells resulted in a 2- to 3-fold increase in PRL content and release. For this study we chose GH3 cells (a somatomammotroph tumor cell line) to determine whether coculturing GH3 with PP cells: 1) stimulates the release and cell content of PRL as compared with GH; 2) increases GH3 cell proliferation; and 3) affects PRL messenger RNA (mRNA) levels. Exp 1. GH3 cells (25,000 cells per well; 25K) were cocultured with PP cells (0K, 12.5K, or 25K) from male rats in serum-free media for 1, 2, 4, and 7 days; hormones were measured by RIA. Coculturing resulted in 5- to 10-fold increases in both media and cell PRL that were linear with time and dependent on the number of PP cells. In contrast, media GH increased only 1.5- to 2-fold, and GH cell content reduced by 75%. Exp 2. GH3, PP, and GH3 + PP cells were cultured for 1, 2, and 4 days and then incubated with [3H]thymidine for 5 h. The incorporation of [3H]thymidine in GH3 cells remained constant over time and showed a small, early increase in cocultures. In contrast, incubation of PP cells alone resulted in a 50- to 60-fold rise in [3H]thymidine incorporation from days 1-4 in culture. Exp 3. Cytoplasmic mRNA was determined by slot blot hybridization with 32P-labeled complementary DNA probes for PRL and GH. After coculturing 25K GH3 cells with 12.5K and 25K PP cells for 4 days, PRL mRNA levels increased 15- and 30-fold, respectively, whereas GH mRNA levels rose less than 2-fold. Neither PRL nor GH mRNA were detected in PP cells.
最近有证据表明，垂体后叶（PP），也称为神经中间叶，调节催乳素（PRL）的释放。我们之前报道过，垂体前叶细胞与PP细胞共培养会导致PRL含量和释放增加2至3倍。在本研究中，我们选择了GH3细胞（一种生长激素催乳素分泌瘤细胞系）来确定GH3细胞与PP细胞共培养是否：1）与单独培养GH3细胞相比，刺激PRL的释放和细胞内含量；2）增加GH3细胞增殖；3）影响PRL信使核糖核酸（mRNA）水平。实验1：将GH3细胞（每孔25,000个细胞；25K）与雄性大鼠的PP细胞（0K、12.5K或25K）在无血清培养基中共培养1、2、4和7天；通过放射免疫分析法测定激素水平。共培养导致培养基和细胞内PRL增加5至10倍，且与时间呈线性关系，并取决于PP细胞的数量。相比之下，培养基中的生长激素（GH）仅增加1.5至2倍，且GH细胞内含量减少75%。实验2：将GH3细胞、PP细胞以及GH3 + PP细胞培养1、2和4天，然后与[3H]胸腺嘧啶核苷孵育5小时。随着时间的推移，GH3细胞中[3H]胸腺嘧啶核苷的掺入量保持恒定，且在共培养中早期有小幅增加。相比之下，单独培养PP细胞在培养第1至4天导致[3H]胸腺嘧啶核苷掺入量增加50至60倍。实验3：通过与用于PRL和GH的32P标记互补DNA探针进行狭缝印迹杂交来测定细胞质mRNA。将25K个GH3细胞与12.5K和25K个PP细胞共培养4天后，PRL mRNA水平分别增加了15倍和30倍，而GH mRNA水平升高不到2倍。在PP细胞中未检测到PRL和GH的mRNA。
1）GH3细胞与PP细胞共培养显著刺激PRL基因表达、合成和释放；2）这种反应对PRL具有特异性，对GH影响较小，且不是由于GH3细胞增殖增加所致；3）我们推测中间叶细胞的一个亚群，可能具有增殖能力，是诱导这些效应的原因。
