Department of Anatomy, Tehran University of Medical Sciences, Tehran, Iran.
Biopreserv Biobank. 2021 Aug;19(4):258-268. doi: 10.1089/bio.2020.0121. Epub 2021 Apr 28.
Cryopreservation of spermatogonial stem cells (SSCs) is a useful method for fertility preservation in preadolescent children suffering from cancer. However, SSCs may become damaged during cryopreservation due to the generation of reactive oxygen species (ROS). For this reason, various antioxidant agents have been used to protect SSCs from cryopreservation-induced damages. Recently, it has been reported that miR-30a-5p has antiapoptotic and antioxidant activity. The aim of this study was to assess the antiapoptotic and antioxidant effects of in frozen-thawed SSCs. To this end, SSCs were isolated from male BALB/C mice (3-6 days old) and cultivated for 14 days. After the detection of optimum concentration, a or with Lipofectamine was transfected into SSCs and, finally, the cell groups were frozen for 1 week. After thawing, different properties, including cell viability (using MTT), colonization of SSCs (number and diameter of colonies), ROS generation (using DCFH-DA assay), levels of malondialdehyde (MDA) and superoxide dismutase (SOD), and gene expression of and BAXBax (using quantitative real-time PCR), were investigated. The transfection of SSCs with before the freezing-thawing process significantly increased the viability, number, and diameter of SSCs colonies. Also, the decreased the levels of ROS production and MDA, but it increased the SOD levels. Moreover, the decreased and increased expression in frozen-thawed SSCs. The transfection of SSCs with the can increase cell viability and antioxidant defense, and it can decrease apoptosis during the freezing-thawing process. If SSC is able to produce spermatozoa after the transfection of miR-30a-5p and the freezing-thawing process, it can be suggested as a promising strategy for the cryopreservation of SSCs in prepubertal boys suffering from cancer.
精原干细胞（SSC）的冷冻保存是保存患有癌症的青春期前儿童生育能力的一种有用方法。然而，由于活性氧（ROS）的产生，SSC 在冷冻保存过程中可能会受到损伤。出于这个原因，已经使用了各种抗氧化剂来保护 SSC 免受冷冻保存引起的损伤。最近，有报道称 miR-30a-5p 具有抗凋亡和抗氧化作用。本研究旨在评估在冷冻-解冻 SSC 中 miR-30a-5p 的抗凋亡和抗氧化作用。为此，从雄性 BALB/C 小鼠（3-6 天大）中分离 SSC 并培养 14 天。在检测到最佳浓度后，将 miR-30a-5p 或 miR-30a-5p 与 Lipofectamine 转染到 SSC 中，最后将细胞组冷冻保存 1 周。解冻后，研究了不同的特性，包括细胞活力（使用 MTT）、SSC 定植（集落数量和直径）、ROS 生成（使用 DCFH-DA 测定法）、丙二醛（MDA）和超氧化物歧化酶（SOD）水平，以及基因表达（使用实时定量 PCR）。在冷冻-解冻过程之前，将 miR-30a-5p 转染到 SSC 中可显著提高 SSC 的活力、数量和集落直径。此外，miR-30a-5p 降低了 ROS 产生和 MDA 水平，但增加了 SOD 水平。此外，miR-30a-5p 降低了冷冻-解冻 SSC 中的 基因表达，增加了 基因表达。将 miR-30a-5p 转染到 SSC 中可以增加细胞活力和抗氧化防御能力，并可以减少冷冻-解冻过程中的细胞凋亡。如果在转染 miR-30a-5p 和冷冻-解冻过程后 SSC 能够产生精子，则可以将其作为一种有前途的策略，用于保存患有癌症的青春期前男孩的 SSC。
