Intraovarian injection of 3D-MSC-EVs-ECM gel significantly improved rat ovarian function after chemotherapy.

IF 4.2 2区医学 Q1 ENDOCRINOLOGY & METABOLISM Reproductive Biology and Endocrinology Pub Date : 2024-10-16 DOI:10.1186/s12958-024-01299-3

Yaping Zhang, Dong Li, Yi Han, Min Wu, Shule Zhang, Huixian Ma, Linghong Liu, Xiuli Ju

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Abstract

Background: Restoring the function of the ovary is important for chemotherapy-induced ovarian failure (COF) patients. Stem cell and extracellular vesicles (EVs) therapy show promise but need further improvement.

Methods: Human umbilical cord mesenchymal stem cells (hUC-MSCs) were primarily cultured and further three-dimensional (3D) cultured using an ultra-low attachment surface method. The expression levels of nutritional cytokines and immunomodulatory and stemness-related genes of 3D-cultured hUC-MSCs were analyzed. EVs were isolated by ultracentrifugation and characterized. Ovaries were decellularized with sodium dodecyl sulfate to obtain extracellular matrix (ECM). Lyophilized EVs from three-dimensional (2D) or 3D hUC-MSCs were mixed with ECM to prepare the 2D/3D-MSC-EVs-ECM gels. The therapeutic effect of the MSC-EVs-ECM gel on cyclophosphamide (CTX) -treated rats was analyzed through various tests. RNA sequencing was used to analyze the expression changes of genes before and after treatment.

Results: After culturing in ultra-low attachment dishes, hUC-MSCs aggregated into spheroids and significantly upregulated the expression levels of immunomodulatory and stemness-related genes. The total EVs yield was also upregulated (5.6-fold) after 3D culture. The cell viability of CTX-treated ovarian granulosa cells (OGCs) was significantly rescued by coculture with the 3D-MSC-EVs-ECM gel. Hormones indicative of ovarian function, AMH, E2, and FSH, were recovered in both the CTX + 2D-MSC-EVs-ECM gel group and the CTX + 3D-MSC-EVs-ECM gel group, while the apoptosis-related protein Bax was significantly downregulated. The 3D-MSC-EVs-ECM gel was more effective than the 2D-MSC-EVs-ECM gel. Significantly differentially expressed genes, such as Hbb-b1, Gpd1, and Sirpa, were detected by RNA sequencing. Hbb-b1 was increased in the ovaries of CTX-treated rats, and this increase was attenuated by injecting the 2D/3D-MSC-EVs-ECM gel. Gpd1 was increased after CTX treatment, and this increase was reversed by the 3D-MSC-EVs-ECM gel. Sirpa was decreased in the ovaries of CTX-treated rats, and this decrease was attenuated by injecting the 3D-MSC-EVs-ECM gel.

Conclusions: Our study demonstrated that the 3D-MSC-EVs-ECM gel is an efficient strategy for the recovery of ovarian function in CTX-induced ovarian failure.

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卵巢内注射三维间充质干细胞-EVs-ECM凝胶可显著改善大鼠化疗后的卵巢功能。

背景：恢复卵巢功能对化疗引起的卵巢功能衰竭（COF）患者非常重要。方法：主要培养人脐带间充质干细胞（hUC-MSCs），并采用超低附着面方法进一步进行三维（3D）培养。分析了三维培养的人脐带间充质干细胞的营养细胞因子、免疫调节基因和干细胞相关基因的表达水平。通过超速离心法分离出EVs并对其进行表征。用十二烷基硫酸钠对卵巢进行脱细胞处理，以获得细胞外基质（ECM）。将来自三维（2D）或三维 hUC-MSCs 的冻干 EVs 与 ECM 混合，制备 2D/3D-MSC-EVs-ECM 凝胶。通过各种测试分析了间充质干细胞-EVs-ECM凝胶对环磷酰胺（CTX）治疗大鼠的治疗效果。采用 RNA 测序分析治疗前后基因表达的变化：结果：在超低附着力培养皿中培养后，hUC-间充质干细胞聚集成球，并显著上调了免疫调节和干性相关基因的表达水平。三维培养后，EVs总产量也有所提高（5.6倍）。与3D-间充质干细胞-EVs-ECM凝胶共培养后，经CTX处理的卵巢颗粒细胞（OGCs）的细胞存活率明显提高。CTX+2D-间充质干细胞-EVs-ECM凝胶组和CTX+3D-间充质干细胞-EVs-ECM凝胶组的卵巢功能指示激素AMH、E2和FSH均得到恢复，而凋亡相关蛋白Bax则明显下调。3D-MSC-EVs-ECM 凝胶比 2D-MSC-EVs-ECM 凝胶更有效。通过 RNA 测序，检测到 Hbb-b1、Gpd1 和 Sirpa 等差异表达基因。经 CTX 处理的大鼠卵巢中 Hbb-b1 增加，而注射 2D/3D-MSC-EVs-ECM 凝胶后，这种增加有所减弱。CTX 处理后，Gpd1 增高，3D-间充质干细胞-EVs-ECM 凝胶可逆转这种增高。经CTX处理的大鼠卵巢中Sirpa减少，而注射3D-间充质干细胞-EVs-ECM凝胶后，Sirpa的减少得到了缓解：我们的研究表明，3D-间充质干细胞-EVs-ECM凝胶是恢复CTX诱导的卵巢功能衰竭大鼠卵巢功能的有效策略。

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来源期刊

Reproductive Biology and Endocrinology 医学-内分泌学与代谢

CiteScore

7.90

自引率

2.30%

发文量

161

审稿时长

4-8 weeks

期刊介绍： Reproductive Biology and Endocrinology publishes and disseminates high-quality results from excellent research in the reproductive sciences. The journal publishes on topics covering gametogenesis, fertilization, early embryonic development, embryo-uterus interaction, reproductive development, pregnancy, uterine biology, endocrinology of reproduction, control of reproduction, reproductive immunology, neuroendocrinology, and veterinary and human reproductive medicine, including all vertebrate species.