Functional and morphological studies on isolated Leydig cells: purification by centrifugal elutriation and metrizamide fractionation.
Functional and morphological studies on isolated Leydig cells: purification by centrifugal elutriation and metrizamide fractionation.
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对分离的 Leydig 细胞的功能和形态学研究：通过离心淘洗和甲曲酰胺分级纯化。
DOI：
10.1210/endo-114-2-499
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发表时间：
1984
期刊：
影响因子：
4.8
通讯作者：
M. Dufau
中科院分区：
文献类型：
--
作者：
D. Aquilano;M. Dufau
通过离心淘洗分离大鼠间质细胞，以促进 Leydig 细胞的纯化，用于体外促性腺激素作用机制的分析。通过此过程，约 1 小时内将来自成年大鼠睾丸的 10(9) 个胶原酶分散的间质细胞分成 12 个部分。级分 1-7（沉降速度，1.9-12 mm/h.g）占所用细胞总数的 80-85%，包括红细胞、淋巴细胞、生发细胞、巨噬细胞、内皮细胞、受损的 Leydig 细胞，并且包含少于 4% 的完整 Leydig 细胞。级分 8-12（沉降速度，大于 12 mm/h.g）包含 15-20% 的原始细胞，并含有 90-95% 的完整 Leydig 细胞。尽管沉降速度不同，但富含 Leydig 细胞的组分的 LH 受体含量相似（平均值 +/- SD，36,115 +/- 4,815 个位点/细胞），并且在睾酮和 cAMP 对 hCG 的反应方面显示出比原始细胞制剂相似的 5 倍增加。合并的富含 Leydig 细胞的级分 (8-12) 在 16-24% Metrizamide 梯度上进一步解析为 5 个条带。带 II-V（密度范围，1.075-1.110 g/ml）含有纯 Leydig 细胞，带 I（1.048 g/ml）含有粗线期精母细胞，这是通过淘洗获得的富含 Leydig 细胞级分中存在的污染细胞类型。 4 个富含 Leydig 细胞的条带均显示出相似的形态和功能活性。使用 14-32% Metrizamide 梯度观察到基本相似的结果。通过 hCG 处理在体内脱敏并通过淘洗分离的 Leydig 细胞也通过 Metrizamide 梯度分解为 4 个条带，但由于最初存在于最重条带中的约 50% 的细胞转移到较低密度部分，因此在梯度中显示出重新分布。然而，尽管密度发生了变化，Leydig 细胞条带在体外仍表现出相似程度的受体下调和类固醇对 hCG 反应的损害。这项研究表明，离心淘析是获得大量纯化（大于 90%）和活性 Leydig 细胞的快速有效方法。在 Metrizamide 梯度中进一步分离富含 Leydig 细胞的组分，可以实现 Leydig 细胞的完全纯化，这是单独通过密度梯度离心无法实现的。由于反应较弱或不活跃的间质细胞表现出不同程度的结构损伤，因此此类细胞不应被视为具有生理意义的群体。（摘要截断为 400 字）
Rat interstitial cells were fractionated by centrifugal elutriation to facilitate the purification of Leydig cells for analysis of mechanisms of gonadotropin action in vitro. By this procedure, 10(9) collagenase-dispersed interstitial cells from adult rat testes were separated into 12 fractions in about 1 h. Fractions 1-7 (sedimentation velocities, 1.9-12 mm/h.g) comprised 80-85% of the total cells applied, including erythrocytes, lymphocytes, germinal cells, macrophages, endothelial cells, damaged Leydig cells, and contained less than 4% intact Leydig cells. Fractions 8-12 (sedimentation velocities, greater than 12 mm/h.g) comprised 15-20% of the original cells and contained 90-95% intact Leydig cells. Despite their different sedimentation velocities, the Leydig cell-rich fractions were similar in their LH receptor content (mean +/- SD, 36,115 +/- 4,815 sites/cell) and showed similar 5-fold increases above the original cell preparation in testosterone and cAMP responses to hCG. The pooled Leydig cell-rich fractions (8-12) were further resolved on 16-24% Metrizamide gradients into 5 bands. Bands II-V (density range, 1.075-1.110 g/ml) contained pure Leydig cells, and band I (1.048 g/ml) contained pachytene spermatocytes, the contaminating cell type present in the Leydig cell-rich fractions obtained by elutriation. Each of the 4 Leydig cell-rich bands showed similar morphology and functional activity. Essentially similar results were observed using 14-32% Metrizamide gradients. Leydig cells desensitized in vivo by hCG treatment and isolated by elutriation were also resolved by Metrizamide gradients into 4 bands, but showed a redistribution in the gradient, due to the shift of about 50% of the cells originally present in the heaviest bands to lower density fractions. However, in spite of their changes in density, the Leydig cell bands still showed similar degrees of receptor down-regulation and impairment of the steroid responses to hCG in vitro. This study has demonstrated that centrifugal elutriation is a rapid and effective method for obtaining large quantities of purified (greater than 90%) and active Leydig cells. Further resolution of the Leydig cell-rich fractions in Metrizamide gradients has allowed complete Leydig cell purification, which is not achieved by density gradient centrifugation alone. Since less responsive or inactive Leydig cells displayed various degrees of structural damage, such cells should not be considered as a population of physiological significance.(ABSTRACT TRUNCATED AT 400 WORDS)
影响因子：
3.6
作者：
MEISTRICH, ML;LONGTIN, J;MACE, ML
通讯作者：
MACE, ML