慢性低度炎症和卵巢生殖细胞（OGSCs）的衰老是卵巢储备功能下降的重要原因，导致卵巢老化和不孕不育。调节慢性炎症有望促进OGSCs的增殖和分化，成为维护和重塑卵巢功能的关键手段。我们先前的研究表明，壳聚糖寡糖（Cos）通过改善免疫相关因子的分泌促进OGSCs的增殖和卵巢功能重塑，但机制尚不明确，卵巢中各种炎症介质的重要来源－巨噬细胞的作用还需要进一步研究。本研究应用巨噬细胞和OGSCs共培养的方法观察Cos对OGSCs的影响和机制，并探究巨噬细胞在其中的重要贡献。我们的发现为预防和治疗早发性卵巢衰竭和不孕不育提供了新的药物治疗选项和方法。我们使用巨噬细胞和OGSCs共培养的方法观察了Cos对OGSCs的影响和机制，探究了巨噬细胞在这一过程中的重要贡献。我们采用免疫组化染色来定位小鼠卵巢中的OGSCs，采用免疫荧光染色、RT-qPCR和ALP染色来鉴定OGSCs，并采用CCK-8和western blot来评估OGSCs的增殖。采用β-半乳糖苷酶（SA-β-Gal）染色和western blot来检测周期蛋白依赖性激酶抑制剂1A（P21）、P53、重组Sirtuin 1（SIRT1）和重组Sirtuin 3（SIRT3）的变化。通过Western blot和ELISA探究免疫因子IL-2、IL-10、TNF-α和TGF-β的水平。我们发现Cos能够剂量和时间依赖性地促进OGSCs的增殖，伴随着IL-2、TNF-α增加和IL-10、TGF-β减少。小鼠单核细胞-巨噬细胞白血病细胞（RAW）也能够产生与Cos相同的效应。当与Cos结合使用时，它会增强Cos在OGSCs中的增殖效应，并进一步增加IL-2、TNF-α并进一步降低IL-10、TGF-β。巨噬细胞能够增强Cos在OGSCs中的增殖效应，这也与IL-2、TNF-α进一步增加和IL-10、TGF-β进一步降低有关。在本研究中，我们确定Cos和RAW分别增加了抗衰老基因SIRT-1和SIRT-3蛋白水平，而衰老相关的SA-β-Gal和衰老基因P21和P53被降低。Cos和RAW在延缓OGSCs的衰老方面具有保护作用。此外，RAW还可以通过Cos降低SA-β-Gal和衰老基因P21和P53，进一步增加OGSCs中的SIRT1和SIRT3蛋白水平。总之，Cos和巨噬细胞通过调节炎性因子在促进OGSCs功能和延缓卵巢衰老方面具有协同作用。©2023年作者。

Chronic low-grade inflammation and ovarian germline stem cells (OGSCs) aging are important reasons for the decline of ovarian reserve function, resulting in ovarian aging and infertility. Regulation of chronic inflammation is expected to promote the proliferation and differentiation of OGSCs, which will become a key means for maintaining and remodeling ovarian function. Our previous study demonstrated that Chitosan Oligosaccharides (Cos) promoted the OGSCs proliferation and remodelled the ovarian function through improving the secretion of immune related factors,but the mechanism remains unclear, and the role of macrophages, the important source of various inflammatory mediators in the ovary needs to be further studied. In this study, we used the method of macrophages and OGSCs co-culture to observe the effect and mechanism of Cos on OGSCs, and explore what contribution macrophages give during this process. Our finding provides new drug treatment options and methods for the prevention and treatment of premature ovarian failure and infertility.We used the method of macrophages and OGSCs co-culture to observe the effect and mechanism of Cos on OGSCs, and explore the important contribution of macrophages in it. The immunohistochemical staining was used to locate the OGSCs in the mouse ovary. Immunofluorescent staining, RT-qPCR and ALP staining were used to identify the OGSCs. CCK-8 and western blot were used to evaluate the OGSCs proliferation. β-galactosidase(SA-β-Gal) staining and western blot were used to detect the changing of cyclin-dependent kinase inhibitor 1A(P21), P53, Recombinant Sirtuin 1(SIRT1) and Recombinant Sirtuin 3(SIRT3). The levels of immune factors IL-2, IL-10, TNF-α and TGF-β were explored by using Western blot and ELISA.We found that Cos promoted OGSCs proliferation in a dose-and time-dependent manner, accompanied by IL-2, TNF-α increase and IL-10, TGF-β decrease. Mouse monocyte-macrophages Leukemia cells(RAW) can also produce the same effect as Cos. When combined with Cos, it can enhance the proliferative effect of Cos in OGSCs, and further increase IL-2, TNF-α and further decrease IL-10, TGF-β. The macrophages can enhance the proliferative effect of Cos in OGSCs is also associated with the further increase in IL-2, TNF-α and the further decrease in IL-10, TGF-β. In this study, we determined that the anti-aging genes SIRT-1 and SIRT-3 protein levels were increased by Cos and RAW respectively, whereas the senescence-associated SA-β-Gal and aging genes P21 and P53 were decreased. Cos and RAW had a protective effect on OGSCs delaying aging. Furthermore, RAW can further decrease the SA-β-Gal and aging genes P21 and P53 by Cos, and further increase SIRT1 and SIRT3 protein levels in OGSCs by Cos.In conclusion, Cos and macrophages have synergistic effects on improving OGSCs function and delaying ovarian aging by regulating inflammatory factors.© 2023. The Author(s).