摘要目的:探讨人脐带间充质干细胞（human umbilical cord mesenchymal stem cells，hUCMSCs）联合高压氧（hyperbaric oxygen，HBO）对模拟高原环境下小鼠卵巢功能损伤的修复作用及作用机制。方法:按照随机数字表法将64只6周龄C57BL/6雌鼠随机分为模型组、hUCMSCs组、hUCMSCs+HBO组和对照组，每组16只。其中模型组、hUCMSCs组、hUCMSCs+HBO组小鼠放置在低压氧舱模拟海拔6 500 m高原环境中持续饲养15 d，构建卵巢功能损伤模型。从造模成功后第1天开始给予对照组与模型组0.2 mL生理盐水尾静脉注射，hUCMSCs组、hUCMSCs+HBO组给予0.2 mL/只hUCMSCs（1×10 6个细胞）尾静脉注射，1次/周，共3次，持续干预15 d，hUCMSCs+HBO组每天予以HBO。记录小鼠一般情况、体质量、动情周期变化、性激素水平、双侧卵巢湿重、卵巢指数、各级卵泡计数情况、卵巢组织病理变化及超微结构改变，Western blotting检测卵巢组织中转化生长因子β1（transforming growth factor beta 1，TGF-β1）、Smad家族成员3（Smad family member 3，Smad3）蛋白的表达，以及各组小鼠产仔数、子代体质量，从而评估各组小鼠生育能力。 结果:①与hUCMSCs组比较，hUCMSCs+HBO组小鼠动情周期、体质量、双侧卵巢湿重、卵巢指数差异均无统计学意义（均 P>0.05）；血清抗苗勒管激素（anti-Müllerian hormone，AMH）、雌二醇、孕酮、卵泡刺激素（follicle-stimulating hormone，FSH）、黄体生成素（luteinizing hormone，LH）水平及各级卵泡计数差异均无统计学意义（均 P>0.05），但hUCMSCs+HBO组卵巢组织颗粒细胞的核膜完整、个别线粒体轻度肿胀、粗面内质网无扩张、高尔基体无肿胀等，TGF-β1、Smad3蛋白表达水平均升高（ P=0.010， P<0.001），子代情况及窝仔数差异均无统计学意义（均 P>0.05）。②与对照组比较，hUCMSCs+HBO组小鼠体质量、双侧卵巢湿重、卵巢指数、血清AMH、雌二醇、孕酮水平略低，血清FSH、LH水平稍偏高，但差异均无统计学意义（均 P>0.05），各级卵泡计数间差异亦均无统计学意义（均 P>0.05），hUCMSCs+HBO组卵巢组织颗粒细胞的核膜、线粒体、粗面内质网、高尔基体等细胞器结构接近于对照组超微结构变化。hUCMSCs+HBO组和对照组TGF-β1蛋白表达差异无统计学意义（ P=0.253）。hUCMSCs+HBO组Smad3蛋白表达水平高于对照组（ P<0.001），子代体质量、窝仔数及生活习性组间差异均无统计学意义（均 P>0.05）。 结论:hUCMSCs联合HBO干预治疗和hUCMSCs的单一治疗均能安全、有效地促进高原环境下受损卵巢组织的卵巢功能修复作用，但hUCMSCs联合HBO修复作用更好。hUCMSCs联合HBO可能是通过上调TGF-β1/Smad3信号通路，修复受损卵巢组织的功能，从而提高缺氧环境下小鼠的生育能力。

abstractsObjective:To investigate the reparative effects and underlying mechanisms of human umbilical cord mesenchymal stem cells (hUCMSCs) in conjunction with hyperbaric oxygen (HBO) on ovarian dysfunction in mice exposed to simulated high-altitude conditions.Methods:A total of 64 six-week-old female C57BL/6 mice were randomly allocated according to random number table, assigned to control group, model group, hUCMSCs group and hUCMSCs+HBO group, with 16 mice in each group. Mice in model group, hUCMSCs group and hUCMSCs+HBO group were placed in a hypobaric oxygen chamber to simulate a high-altitude environment at an elevation of 6 500 m for 15 d, thereby establishing a model of ovarian dysfunction. Beginning on the first day following model establishment, the control and model groups received intravenous injections of 0.2 mL saline via the tail vein. In contrast, the hUCMSCs group and the hUCMSCs+HBO group received 0.2 mL hUCMSCs (1×10 6 cells) per mouse through tail vein injection, administered once a week for three weeks, with continuous intervention lasting 15 d. Furthermore, the hUCMSCs+HBO group were subjected to daily hyperbaric oxygen treatment. The recorded variables included general health status, body weight, estrous cycle changes, serum hormone levels, bilateral ovarian wet weight, ovarian index, follicular development assessment, pathological alterations of ovarian tissue, ultrastructural changes, and the expression levels of transforming growth factor beta 1 (TGF-β1) and Smad family member 3 (Smad3) detected by Western blotting in ovarian tissue. Additionally, litter size and offspring body weight were measured across all groups to evaluate the reproductive capacity of the mice. Results:1) Compared with the hUCMSCs group, the hUCMSCs+HBO group exhibited no statistically significant differences in estrous cycle, body weight, bilateral ovarian wet weight, or ovarian index (all P>0.05). Furthermore, serum levels of anti-Müllerian hormone (AMH), estradiol, progesterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH), as well as counts of various stages of follicles in hUCMSCs+HBO group, did not demonstrate statistically significant differences compared with hUCMSCs group (all P>0.05). Notably, in the hUCMSCs+HBO group, the integrity of the nuclear membrane in granulosa cells of the ovarian tissue was preserved, with only mild swelling observed in individual mitochondria, while no expansion of the rough endoplasmic reticulum or swelling of the Golgi apparatus were noted. Additionally, expression levels of TGF-β1 and Smad3 proteins in the hUCMSCs+HBO group were significantly elevated compared with the hUCMSCs group ( P=0.010, P<0.001). The conditions of the offspring and litter size showed no statistically significant differences between the two groups (all P>0.05). 2)Compared with control group, the hUCMSCs+HBO group had slightly lower values for body weight, bilateral ovarian wet weight, ovarian index, serum levels of AMH, estradiol, and progesterone, while had slightly higher serum levels of FSH and LH without statistically significant differences (all P>0.05). Importantly, the ultrastructural characteristics of granulosa cells in the ovarian tissue of the hUCMSCs+HBO group closely resembled those of control group, displaying intact structures of the nuclear membrane, mitochondria, rough endoplasmic reticulum, and Golgi apparatus. There was no significant difference in TGF-β1 protein level between hUCMSCs+HBO group and control group ( P=0.253), but Smad3 protein level in hUCMSCs+HBO group was higher than that in control group ( P<0.001). No statistically significant differences were detected in offspring body weight, litter size, or behavioral tendencies (all P>0.05). Conclusion:Both hUCMSCs and the combination of hUCMSCs with HBO intervention demonstrated a safe and effective promotion of functional repair in damaged ovarian tissue under hypoxic conditions. Notably, the combination treatment of hUCMSCs with HBO exhibited a synergistic effect compared with hUCMSCs alone. The potential mechanism underlying this enhanced functional repair may involve the upregulation of the TGF-β1/Smad3 signaling pathway, which could ultimately improve fertility outcomes in mice subjected to hypoxic environments.