Oosterom R, Verleun T, Lamberts S W
Mol Cell Endocrinol. 1983 Feb;29(2):197-212. doi: 10.1016/0303-7207(83)90213-7.
Culture conditions for rat pituitary cells were investigated which would result in high PRL synthesis and secretion with maintenance of dopamine-mediated inhibition of PRL secretion. From five commercially available media, RPMI resulted in the highest PRL content and secretion, but no inhibition of PRL secretion by dopamine was observed. MEM with Earle's salts fulfilled best our requirements for culturing functional PRL-secreting cells. PRL secretion was not affected by variations in the concentration of fetal calf serum, but was positively correlated with increasing horse serum concentrations. TRH-induced PRL release increased with increasing serum concentrations and was positively correlated with the concentration of 17 beta-estradiol in the culture medium (P less than 0.0025). An increase in the sodium bicarbonate concentration from 0.85 to 3.0 g/l resulted in a 4-fold stimulation of PRL synthesis and in a 27-fold stimulation of PRL secretion. However, at bicarbonate concentrations above 2.6 g/l, inhibition of PRL secretion by 500 nM dopamine was lost. The addition of 20 mM Hepes to the culture medium decreased basal PRL secretion by 48 +/- 13% (P less than 0.01), while dopamine inhibition of PRL secretion was reduced from 49 +/- 10% to 24 +/- 8% (P less than 0.05). When an increasing number of pituitary cells was cultured in a constant volume, PRL secretion expressed per cell increased up to 0.3-0.4 X 10(6) cells/dish/2 ml. With higher cell concentrations of up to 1 X 10(6) cells/dish, PRL secretion per cell diminished significantly, which indicates a direct negative feedback of high medium PRL on the PRL-secreting pituitary cells. In this culture system dopamine inhibited PRL secretion over a 4 h period in a dose-dependent manner (IC50 20 nM), while no paradoxical stimulation of PRL secretion was observed with low dopamine concentrations. However, a 25% stimulation (P less than 0.05) of PRL secretion by 0.1 nM dopamine could be obtained by addition of 0.01% ascorbic acid, which by itself decreased basal PRL secretion by 49% (P less than 0.01). Thus, tissue culture conditions that result in high PRL production are not necessarily the best choice, since dopamine-mediated inhibition of PRL secretion is another important parameter for the functioning of lactotrophs in culture. The best compromise is MEM with 2.2 g/l of sodium bicarbonate, without Hepes buffer and supplemented with 10% FCS.
对大鼠垂体细胞的培养条件进行了研究，旨在实现高催乳素（PRL）合成与分泌，同时维持多巴胺介导的对PRL分泌的抑制作用。在五种市售培养基中，RPMI培养基中的PRL含量和分泌量最高，但未观察到多巴胺对PRL分泌的抑制作用。含Earle氏盐的MEM培养基最符合我们培养功能性PRL分泌细胞的要求。PRL分泌不受胎牛血清浓度变化的影响，但与马血清浓度的增加呈正相关。促甲状腺激素（TRH）诱导的PRL释放随血清浓度增加而增加，且与培养基中17β-雌二醇的浓度呈正相关（P<0.0025）。碳酸氢钠浓度从0.85g/L增加到3.0g/L，导致PRL合成增加4倍，PRL分泌增加27倍。然而，当碳酸氢钠浓度高于2.6g/L时，500nM多巴胺对PRL分泌的抑制作用消失。向培养基中添加20mM羟乙基哌嗪乙磺酸（Hepes）可使基础PRL分泌降低48±13%（P<0.01），而多巴胺对PRL分泌的抑制作用从49±10%降低至24±8%（P<0.05）。当在恒定体积中培养的垂体细胞数量增加时，每个细胞的PRL分泌量增加，直至达到0.3 - 0.4×10⁶个细胞/培养皿/2ml。当细胞浓度高达1×10⁶个细胞/培养皿时，每个细胞的PRL分泌量显著减少，这表明培养基中高浓度的PRL对PRL分泌性垂体细胞存在直接负反馈作用。在该培养系统中，多巴胺在4小时内以剂量依赖性方式抑制PRL分泌（半数抑制浓度[IC50]为20nM），低浓度多巴胺未观察到对PRL分泌的反常刺激作用。然而，添加0.01%抗坏血酸可使0.1nM多巴胺刺激PRL分泌25%（P<0.05），而抗坏血酸本身可使基础PRL分泌降低49%（P<0.01）。因此，导致高PRL产生的组织培养条件不一定是最佳选择，因为多巴胺介导的对PRL分泌的抑制作用是培养中催乳细胞功能的另一个重要参数。最佳折衷方案是使用含2.2g/L碳酸氢钠、不含Hepes缓冲液并添加10%胎牛血清（FCS）的MEM培养基。
