Chamras H, Hershman J M, Stanley T M
Endocrinology. 1984 Oct;115(4):1406-11. doi: 10.1210/endo-115-4-1406.
To study the metabolism of thyrotrophs and dynamics of TSH secretion in vitro, it is desirable to have a highly enriched population of thyrotrophs. For that purpose, centrifugal elutriation, a recently developed cell isolation method based on the size and density of cells, was used to prepare thyrotrophs from a cell suspension of adult male rat pituitary cells. Trypsin-dispersed cells (4-8 X 10(7] were loaded into the elutriation rotor (Beckman, JE-6) operating at 2800 rpm. Twelve cell fractions were collected at variable rotor speed (2000-2800 rpm) and increasing medium flow rate (10-103 ml/min). Cell recovery was 77-98%. The viability of the cells after elutriation was 90-95% based on trypan blue exclusion. Each fraction was analyzed for TSH, GH, and PRL content and for TRH-stimulated TSH release by RIA. Thyrotrophs were found predominantly in fractions 8-11 (flow rate 38-75 ml/min) based on TSH RIA. The mean TSH concentration in these fractions was 56 +/- 13.6 (+/- SD) microU/10(3) cells compared with 7.6 +/- 3.8 microU/10(3) cells in the initial cell suspension, representing a 7- to 8-fold enrichment of the thyrotrophs. Incubation with 20 nM TRH for 3 h increased the TSH release of cells eluted in fractions 8-11 by 3- to 5-fold; there was no significant increase in TSH release in fractions 3-6. Centrifugal elutriation may be used to prepare a uniform highly enriched thyrotroph fraction with excellent recovery from a suspension of rat pituitary cells. This technique should be valuable for study of the metabolism of thyrotrophs.
为了在体外研究促甲状腺激素细胞的代谢及促甲状腺激素（TSH）分泌的动态变化，需要获得高度富集的促甲状腺激素细胞群体。为此，采用了离心淘洗法，这是一种基于细胞大小和密度的新近开发的细胞分离方法，用于从成年雄性大鼠垂体细胞悬液中制备促甲状腺激素细胞。将胰蛋白酶分散的细胞（4 - 8×10⁷）装入以2800转/分钟运行的淘洗转子（贝克曼，JE - 6）中。在可变的转子速度（2000 - 2800转/分钟）和增加的培养基流速（10 - 103毫升/分钟）下收集12个细胞组分。细胞回收率为77 - 98%。基于台盼蓝排斥法，淘洗后细胞的活力为90 - 95%。通过放射免疫分析（RIA）对每个组分的TSH、生长激素（GH）和催乳素（PRL）含量以及TRH刺激的TSH释放进行分析。基于TSH RIA，促甲状腺激素细胞主要存在于第8 - 11组分（流速38 - 75毫升/分钟）中。这些组分中TSH的平均浓度为56±13.6（±标准差）微单位/10³个细胞，而初始细胞悬液中为7.6±3.8微单位/10³个细胞，这表明促甲状腺激素细胞富集了7至8倍。用20纳摩尔TRH孵育3小时，可使第8 - 11组分中洗脱的细胞的TSH释放增加3至5倍；第3 - 6组分中TSH释放没有显著增加。离心淘洗法可用于从大鼠垂体细胞悬液中制备均匀的高度富集的促甲状腺激素细胞组分，且回收率良好。该技术对于研究促甲状腺激素细胞的代谢应该是有价值的。
