褪黑素对三维软琼脂培养体系中原代精原干细胞定植的影响。

The effects of melatonin on colonization of neonate spermatogonial mouse stem cells in a three-dimensional soft agar culture system.

机构信息

Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.

Laboratory of Reproductive Endocrinology, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Kagamiyama 1-4-4, Hiroshima, 739-8528, Japan.

出版信息

Stem Cell Res Ther. 2017 Oct 17;8(1):233. doi: 10.1186/s13287-017-0687-y.

DOI:10.1186/s13287-017-0687-y

PMID:29041987

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5646105/

Abstract

BACKGROUND

Melatonin is a pleiotropic hormone with powerful antioxidant activity both in vivo and in vitro. The present study aimed to investigate the effects of melatonin on the proliferation efficiency of neonatal mouse spermatogonial stem cells (SSCs) using a three-dimensional soft agar culture system (SACS) which has the capacity to induce development of SSCs similar to in vivo conditions.

METHODS

SSCs were isolated from testes of neonate mice and their purities were assessed by flow cytometry using PLZF antibody. Isolated testicular cells were cultured in the upper layer of the SACS in αMEM medium in the absence or presence of melatonin extract for 4 weeks.

RESULTS

The identity of colonies was confirmed by alkaline phosphatase staining and immunocytochemistry using PLZF and α6 integrin antibodies. The number and diameter of colonies of SSCs in the upper layer were evaluated at days 14 and 28 of culture. The number and diameter of colonies of SSCs were significantly higher in the melatonin group compared with the control group. The levels of expression of ID-4 and Plzf, unlike c-kit, were significantly higher in the melatonin group than in the control group.

CONCLUSIONS

Results of the present study show that supplementation of the culture medium (SACS) with 100 μM melatonin significantly decreased reactive oxygen species (ROS) production in the treated group compared with the control group, and increased SSC proliferation.

摘要

背景

褪黑素是一种具有强大抗氧化活性的多功能激素，无论是在体内还是体外。本研究旨在使用三维软琼脂培养系统（SACS）研究褪黑素对新生小鼠精原干细胞（SSC）增殖效率的影响，该系统具有诱导 SSC 发育的能力，类似于体内条件。

方法

从新生小鼠睾丸中分离 SSC，并使用 PLZF 抗体通过流式细胞术评估其纯度。将分离的睾丸细胞在不含或含褪黑素提取物的αMEM 培养基中在上层 SACS 中培养 4 周。

结果

通过碱性磷酸酶染色和使用 PLZF 和 α6 整合素抗体的免疫细胞化学证实了集落的同一性。在培养的第 14 天和第 28 天评估了上层 SSC 集落的数量和直径。与对照组相比，褪黑素组的集落数量和直径明显更高。与对照组相比，ID-4 和 Plzf 的表达水平（不同于 c-kit）在褪黑素组中明显更高。