Noghani Amirhessam Eskafi, Asadpour Reza, Saberivand Adel, Mazaheri Zohreh, Hamidian Gholamreza
Department of Clinical Science, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran.
Basic Medical Science Research Center, Histogenotech Company, Tehran, Iran.
Mol Biol Rep. 2022 Mar;49(3):2197-2207. doi: 10.1007/s11033-021-07041-1. Epub 2022 Jan 9.
The main purpose of this study was to investigate the effect of D-serine (DS) and Dizocilpine (MK-801) on the proliferation of spermatogonial stem cells (SSCs) in two-dimensional (2D) and three-dimensional (3D) culture systems.
The SSCs of male NMRI mice were isolated by enzymatic digestion and cultured for two weeks. Then, the identity of SSCs was validated by anti-Plzf and anti-GFR-α1 antibodies via immunocytochemistry (ICC). The proliferation capacity of SSCs was evaluated by their culture on a layer of the decellularized testicular matrix (DTM) prepared from mouse testis, as well as two-dimensional (2D) with different mediums. After two weeks of the initiation of proliferation culture on 3D and 2D medium, the pre-meiotic at the mRNA and protein levels were evaluated via qRT-PCR and flow cytometry methods, respectively. The results showed that the proliferation rate of SSCs in 3D culture with 50 mM glutamic acid and 20 mM D-serine was significantly different from other groups after 14 days treatment. mRNA expression levels of promyelocytic leukemia zinc finger (Plzf) in 3D cultures supplemented by 20 mM D-serine and 50 mM glutamic acid were considerably higher than the 3D control group (p < 0.001). The flow cytometry analysis revealed that the amount of Plzf in the 2D-culture groups of SSCs with 20 mM MK-801 was considerably lower compared to the 2D-culture control group (p < 0.001).
This study indicated that decellularized testicular matrix supplemented with D-serine and glutamic acid could be considered a promising vehicle to support cells and provide an appropriate niche for the proliferation of SSCs.
本研究的主要目的是探讨D-丝氨酸（DS）和地卓西平（MK-801）对二维（2D）和三维（3D）培养系统中精原干细胞（SSCs）增殖的影响。
通过酶消化法分离雄性NMRI小鼠的SSCs，并培养两周。然后，通过免疫细胞化学（ICC）用抗Plzf和抗GFR-α1抗体验证SSCs的身份。通过将SSCs培养在由小鼠睾丸制备的脱细胞睾丸基质（DTM）层以及含有不同培养基的二维（2D）环境中，评估SSCs的增殖能力。在3D和2D培养基上开始增殖培养两周后，分别通过qRT-PCR和流式细胞术方法评估减数分裂前细胞在mRNA和蛋白质水平的情况。结果显示，在14天的处理后，添加50 mM谷氨酸和20 mM D-丝氨酸的3D培养体系中SSCs的增殖率与其他组有显著差异。添加20 mM D-丝氨酸和50 mM谷氨酸的3D培养体系中早幼粒细胞白血病锌指蛋白（Plzf）的mRNA表达水平显著高于3D对照组（p < 0.001）。流式细胞术分析显示，与2D培养对照组相比，添加20 mM MK-801的2D培养组中SSCs的Plzf含量显著降低（p < 0.001）。
本研究表明，添加D-丝氨酸和谷氨酸的脱细胞睾丸基质可被视为一种有前景的载体，用于支持细胞并为SSCs的增殖提供合适的微环境。
