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孕酮促进小鼠胚胎干细胞的增殖和存活能力

申善维, 宋后燕*

复旦大学上海医学院教育部分子医学重点实验室，上海 200032

摘要

已有研究表明雌二醇刺激小鼠胚胎干细胞(mouse embryonic stem cells, mES cells)的增殖，为了探讨另一种甾体激素— 孕酮对mES 细胞增殖的影响，本研究分别采用MTT 实验和BrdU 掺入实验获得增殖曲线，克隆形成实验(clone forming assay) 检测孕酮对单细胞存活能力和增殖能力的影响，Western blot 检测胚胎干细胞特异的标志基因Oct-4 的表达，流式细胞术 (fluorescence activated cell sorting, FACS)检测细胞周期，以及real-time PCR 检测细胞周期有关的基因和部分原癌基因的表达。 结果显示，和雌激素一样，孕酮也具有促mES 细胞增殖的作用。在孕酮处理组，克隆数目比对照组显著增多。Western blot 结果显示在孕酮处理组，Oct-4 表达变化不大，mES 仍处在干细胞状态。FACS 结果显示：加入孕酮后，和对照组相比， mES 处在S+G2/M 期的细胞比例增多。Real-time PCR 结果显示孕酮处理使细胞周期蛋白(cyclins)、细胞周期蛋白依赖的蛋白激 酶(cyclin-dependent kinases)、c-myc、c-fos 基因表达上调。以上结果提示，孕酮促进mES 细胞增殖和存活能力，孕酮促增 殖的作用可能和上述细胞周期有关基因的表达上调有关。

关键词： 孕酮; 胚胎干细胞; 细胞周期蛋白; 细胞周期蛋白依赖的蛋白激酶; 原癌基因

分类号：Q548+.2

Progesterone promotes propagation and viability of mouse embryonic stem cells

SHEN Shan-Wei, SONG Hou-Yan

The Key Laboratory of Molecular Medicine, Ministry of Education, Shanghai Medical School, Fudan University, Shanghai 200032, China

Abstract

It has been known that estrogen-17β stimulates proliferation of mouse embryonic stem (mES) cells. To explore the function of another steroid hormone progesterone, we used MTT method and BrdU incorporation assay to obtain growth curves, clone forming assay to detect the propagation and viability of individual mES cells, Western blot to test the expression of ES cell marker gene Oct-4, fluorescence activated cell sorter (FACS) to test cell cycle, and real-time PCR to detect the expressions of cyclins, cyclin-dependent kinases and proto-oncogenes. The results showed that progesterone promoted proliferation of mES cells. The number of clones was more in progesterone-treated group than that in the control group. The expression of pluripotency-associated transcriptional factor Oct-4 changed little after progesterone treatment as shown by Western blot, indicating that most of mES cells were in undifferentiated state. The results of FACS proved that progesterone promoted DNA synthesis in mES cells. The proportion of mES cells in S+G2/M phase was higher in progesterone-treated group than that in the control group. Cyclins and cyclin-dependent kinases, as well as protooncogenes (c-myc, c-fos) were up-regulated when cells were treated with progesterone. The results obtained indicate that progesterone promotes propagation and viability of mES cells. The up-regulation of cell cycle-related factors might contribute to the function of progesterone.

Key words: Progesterone; embryonic stem cells; cyclins; cyclin-dependent kinases; proto-oncogenes

收稿日期：2009-04-16　　录用日期：2009-08-04

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引用本文：

申善维, 宋后燕*. 孕酮促进小鼠胚胎干细胞的增殖和存活能力[J]. 生理学报 2009; 61 (5): 431-438.

SHEN Shan-Wei, SONG Hou-Yan. Progesterone promotes propagation and viability of mouse embryonic stem cells. Acta Physiol Sin 2009; 61 (5): 431-438 (in Chinese with English abstract).