BMSCs-Exos对环磷酰胺诱导的大鼠精原细胞损伤的修复作用

Repair Effect of BMSCs-Exos on Cyclophosphamide-Induced Rat Germ Cell Injury

摘要: 目的：非阻塞性无精子症(NOA)是男性不孕症的严重问题，目前医学上缺乏有效的治疗方法。骨髓来源的间充质干细胞(BMSCs)具有多能分化能力和旁分泌效应，并参与组织修复和再生。本研究旨在探讨BMSCs外泌体(BMSCs-exos)对环磷酰胺诱导的精原细胞凋亡的抑制和改善细胞增殖的作用，为临床上应用间充质干细胞外泌体治疗细胞化疗损伤提供前期实验基础，并为后续进一步研究其机制奠定前期基础。方法：取大鼠股骨骨髓，使用流式细胞术培养，并使用成骨及成脂诱导培养鉴定BMSCs。然后使用超速离心法获取并收集BMSC细胞外泌体。通过透射电子显微镜、Western blotting和纳米流式检测蛋白标记分析鉴定BMSCs-exos。将大鼠处死后收集培养生精细胞，然后进行瑞氏–姬姆萨染色证实培养的细胞确为精原细胞，将精原细胞经过不同浓度的环磷酰胺(CTX)的处理，CCK8筛选出最佳CTX处理浓度12 uM，然后再将精原细胞进行分组处理：精原细胞组正常培养(Control对照组)、化疗损伤组(CTX组)和化疗损伤 + 外泌体干预组(CTX + BMSCs-exosomes组)，之后对3组细胞进行增殖和凋亡检测。结果：对3组细胞进行的细胞凋亡率检测结果显示，细胞凋亡率：CTX组 > CTX + BMSCs-exosomes组 > Control对照组；细胞增殖能力：Control对照组 > CTX + BMSCs-exosomes组 > CTX组。结论：CTX化疗药物可以增加大鼠生精细胞的凋亡，BMSCs-exos可以抑制大鼠生精细胞的凋亡，增强细胞的增殖。该研究为临床上无精症的治疗提供一定的参考，也为临床上应用间充质干细胞外泌体治疗细胞化疗损伤提供前期实验基础，并为后续进一步研究其机制奠定前期基础。

Abstract: Objective: Non-obstructive azoospermia (NOA) is a serious problem in male infertility, and there is currently no effective treatment in medicine. Bone marrow-derived mesenchymal stem cells (BMSCs) have multipotent differentiation ability and paracrine effects, and participate in tissue re-pair and regeneration. This study aims to explore the inhibitory effect of BMSCs-exosomes on cy-clophosphamide-induced germ cell apoptosis and the improvement of cell proliferation, providing a preliminary experimental basis for the clinical application of mesenchymal stem cell exosomes in the treatment of chemotherapy-induced cell damage, and laying a preliminary foundation for fur-ther research on its mechanism. Methods: Rat bone marrow was taken, cultured using flow cytome-try, and identified as BMSCs using osteogenic and adipogenic induction culture. Then, ultracentrif-ugation was used to obtain and collect BMSC extracellular vesicles. BMSCs-exos were identified by transmission electron microscopy, Western blotting, and nanoflow cytometry protein marker anal-ysis. After rats were killed, cultured germ cells were collected and confirmed by Rhys-Jones staining to be germ cells. The germ cells were treated with different concentrations of cyclophosphamide (CTX), and the optimal CTX treatment concentration was screened by CCK8 as 12 uM. Then the germ cells were divided into three groups: normal culture (Control group), chemotherapy damage group (CTX group), and chemotherapy damage + exosome intervention group (CTX + BMSCs-exosomes group). The proliferation and apoptosis of the three groups of cells were detected. Results: The re-sults of cell apoptosis rate detection of the three groups of cells showed that the cell apoptosis rate was CTX group > CTX + BMSCs-exosomes group > Control group; Cell proliferation ability: Control group > CTX + BMSCs-exosomes group > CTX group. Conclusion: Cyclophosphamide chemotherapy drugs can increase the apoptosis of rat germ cells. BMSCs-exos can inhibit the apoptosis of rat germ cells and enhance cell proliferation. This study provides a certain reference for the treatment of azoospermia in clinical practice, provides a preliminary experimental basis for the clinical applica-tion of mesenchymal stem cell exosomes in the treatment of chemotherapy-induced cell damage, and lays a preliminary foundation for further research on its mechanism.