Isolation of thecal cells: an assessment of purity and steroidogenic potential
Isolation of thecal cells: an assessment of purity and steroidogenic potential
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DOI：
10.1016/s0165-022x(00)00107-x
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发表时间：
2000-09-11
影响因子：
--
通讯作者：
Hearn, MTW
中科院分区：
文献类型：
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作者：
Li, SKB;Hearn, MTW
采用Percoll密度梯度离心法制备的大鼠卵泡膜细胞，在无血清培养条件下培养，观察其对不同浓度卵泡素(FSH)、碱性成纤维细胞生长因子(BFGF)和促黄体生成素(Lh)的反应性。同时测定细胞培养液中雌二醇(E-2)和孕酮(P-4)的含量。结果证实，用这种方法制备和培养的大鼠卵泡膜细胞经不同处理后的细胞培养液中未检测到E-2，因此未见颗粒细胞对膜细胞制剂的污染。在无血清培养条件下，观察了成纤维细胞生长因子-2和黄体生成素对这些细胞的类固醇合成和细胞分化特性的影响。经Percoll纯化的大鼠卵泡膜细胞经不同处理后，经FSH和/或FGF2处理后，P-4的产生与基础细胞培养无差异，同一培养液中未检测到E-2。相反，在无血清细胞培养液中，促黄体生成素(20或50 ng/ml)可促进P-4的产生(P<；0.05)。促黄体生成素浓度越高(50 ng/ml)，对P-4产生的刺激作用越强(P<；0.05)。同时给予促黄体生成素(20或50 ng/ml)和成纤维细胞生长因子-2(1-100 ng/ml)，显示成纤维细胞生长因子-2抑制促黄体生成素刺激的膜细胞产生P_1(P<；0.05)。当促黄体生成素浓度较低时(促黄体生成素-20 ng/ml时EC50为1 ng/ml，促黄体生成素-50 ng/ml时为1 ng/ml)，成纤维细胞生长因子-2的抑制作用更强。因此，本研究的结果表明，通过这种优化的Percoll密度离心法分离的大鼠卵泡膜细胞保持了非常高的类固醇生成潜力和特异性。与没有污染的颗粒细胞一致，这些大鼠卵泡膜细胞制剂在E-2产生方面对FSH处理没有反应。然而，这些大鼠卵泡膜细胞制剂可以在黄体生成素的刺激下，在无血清培养中表达其分化状态，并对生长因子如成纤维细胞生长因子-2做出反应。(C)2000年爱思唯尔科学公司。版权所有。
In the present investigation, the responsiveness of rat thecal cells, prepared by means of an optimised discontinuous Percoll density gradient centrifugation procedure and cultured under serum-free cell culture conditions, to different concentrations of follitropin (FSH), basic fibroblast growth factor (FGF-2 or bFGF), and lutropin (LH) has been examined. The estradiol (E-2) and progesterone (P-4) contents of the cell culture medium were simultaneously determined with aliquots collected after different times of exposure to these regulatory proteins, either individually or in combination. The results confirm that no E-2 could be detected in the cell culture medium of the rat thecal cells prepared and cultured in this manner following all of these different treatments, and hence no contamination of the thecal cell preparations by granulosa cells was evident. The effects of FGF-2 and LH on the steroidogenic and cytodifferentiational properties of these rat thecal cells under serum-free cell culture procedures were also examined. The production of P-4 in the Percoll-purified rat thecal cell cultures receiving different treatments of FSH, and/or FGF-2 did not differ from the basal cell cultures, and no E-2, was detected from the same culture media. In contrast, LH (20 or 50 ng/ml) was found to enhance the production of P-4 (P < 0.05) in the serum-free cell culture media. The stimulation of P-4 production was greater at higher LH concentration (50 ng/ml) (P < 0.05). Concurrent treatment of LH (20 or 50 ng/ml) and FGF-2 (1-100 ng/ml) showed that FGF-2 inhibited the production of P, by LH-stimulated thecal cell cultures (P < 0.05). The inhibition by FGF-2 was greater when LH was at a lower concentration (EC50 < 1 ng/ml at LH-20 ng/ml vs. EC50 > 1 ng/ml at LH-50 ng/ml). The results of the present study thus indicate that rat thecal cells isolated by this optimised Percoll density centrifugation procedure maintain a very high steroidogenic potential and specificity. Consistent with the absence of contaminating, granulosa cells, these rat theca cell preparations do not respond to FSH treatment in terms of E-2 production. However, these rat theca cell preparations can be stimulated by LH to express their differentiated status in serum-free medium and respond to growth factors such as FGF-2. (C) 2000 Elsevier Science BN. AII rights reserved.