低强度超声处理后从小鼠精原干细胞中诱导的胚胎干细胞样细胞的鉴定。

Characterization of embryonic stem-like cells derived from mouse spermatogonial stem cells following low-intensity ultrasound treatment.

机构信息

Anatomical Sciences Department, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.

Institute of Regenerative Medicine, School of Medicine, Wake Forest University, North Carolina, USA; Para-Clinic Department, Medicine Faculty, Kateb University, Kabul, Afghanistan.

出版信息

Rev Int Androl. 2021 Oct-Dec;19(4):264-271. doi: 10.1016/j.androl.2020.05.010. Epub 2020 Dec 23.

DOI:10.1016/j.androl.2020.05.010

PMID:33358310

Abstract

OBJECTIVE

Spermatogonial stem cells (SSCs) are able to form embryonic stem-like cells (ES-like cells) and embryonic bodies (EBs). Low-intensity ultrasound stimulation (LIUS) has positive effects on the growth and differentiation of the different cells. In this study, we tried to investigate the effects of LIUS on SSC differentiation to ES-like cells.

MATERIALS AND METHODS

SSCs were isolated from neonatal mice and their identification was confirmed by tracking of PLZF, Oct-4, and C-Kit proteins. The SSCs and Sertoli cells were co-cultured in DMEM/F12 supplemented with 15% FBS and LIF. SSCs stimulated by LIUS with 200mW/CM intensity. Characterization of obtained ES-like cells was confirmed with Sox2, Oct-4, and SSEA-1 immunofluorescence staining. Also, real-time PCR was performed to analyse the expression of c-Myc and Nanog genes in ES-Like Cells and Stra8, Piwil2 and Plzf genes in SSCs after 21 days of the in vitro culture.

RESULTS

Our results showed c-Kit, PLZF and Oct-4 proteins were expressed positively in SSCs and Sox2, Oct-4, SSEA-1 in the ES-like cells by immunocytochemistry. The results of flow cytometry showed a significant increase in expression of c-Myc and Nanog in ES-like cells compared to SSCs (p<.05), whereas the Stra8, Piwil2, and Plzf became down-regulated during 21 days of culture. ES-like markers cell SSEA-1, Sox2 and Oct-4 were increased in the LIUS group compared to the control group (p<.05).

CONCLUSION

The results indicated that ES-like cells with pluripotency characteristics were derived from SSCs.

摘要

目的

精原干细胞（SSCs）能够形成胚胎干细胞样细胞（ES 样细胞）和胚胎体（EBs）。低强度超声刺激（LIUS）对不同细胞的生长和分化有积极影响。在这项研究中，我们试图研究 LIUS 对 SSC 向 ES 样细胞分化的影响。

材料和方法

从新生小鼠中分离 SSCs，并通过追踪 PLZF、Oct-4 和 C-Kit 蛋白来确认其鉴定。将 SSCs 和支持细胞在补充有 15% FBS 和 LIF 的 DMEM/F12 中共同培养。用 200mW/CM 强度的 LIUS 刺激 SSCs。通过 Sox2、Oct-4 和 SSEA-1 免疫荧光染色确认获得的 ES 样细胞的特征。此外，还进行了实时 PCR 分析，以分析 ES 样细胞中 c-Myc 和 Nanog 基因的表达以及体外培养 21 天后 SSCs 中 Stra8、Piwil2 和 Plzf 基因的表达。

结果

我们的结果表明，免疫细胞化学显示 SSCs 中表达了 c-Kit、PLZF 和 Oct-4 蛋白，ES 样细胞中表达了 Sox2、Oct-4 和 SSEA-1。流式细胞术结果表明，与 SSCs 相比，ES 样细胞中 c-Myc 和 Nanog 的表达显著增加（p<.05），而 Stra8、Piwil2 和 Plzf 在培养 21 天后下调。与对照组相比，LIUS 组 ES 样细胞标志物 SSEA-1、Sox2 和 Oct-4 的表达增加（p<.05）。