中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (7): 985-991.doi: 10.12307/2022.133

• 骨髓干细胞 bone marrow stem cells •    下一篇

骨髓间充质干细胞调控猕猴卵巢的衰老

田  川1，2，朱向情2，杨再玲1，2，鄢东海2，李  晔2，王严影2，杨育坤2，何  洁2，吕冠柯2，蔡学敏2，舒丽萍1，何志旭3，潘兴华1，2   

  1. 1贵州医科大学，组织工程与干细胞实验中心，贵州省贵阳市  550004；2解放军联勤保障部队第920 医院基础医学实验室，云南省细胞治疗技术转化医学重点实验室，干细胞与免疫细胞生物医药技术国家地方联合工程实验室，云南省昆明市  650032；3遵义医科大学附属医院儿科学教研室，贵州省遵义市  563100
  • 收稿日期:2020-09-30 修回日期:2020-10-12 接受日期:2020-11-13 出版日期:2022-03-08 发布日期:2021-10-28
  • 通讯作者: 潘兴华，博士，主任医师，贵州医科大学，组织工程与干细胞实验中心，贵州省贵阳市 550004；解放军联勤保障部队第920 医院基础医学实验室，云南省细胞治疗技术转化医学重点实验室，干细胞与免疫细胞生物医药技术国家地方联合工程实验室，云南省昆明市 650032
  • 作者简介:田川，男，1994 年生，贵州省遵义市人，土家族，贵州医科大学在读硕士，主要从事骨髓间充质干细胞抗衰老研究。
  • 基金资助:
    云南省科技计划项目重大科技专项(2018ZF007)，项目负责人：潘兴华；医院“申请-审核制”专项项目(2019YGCO2)，项目负责人：朱向情

Bone marrow mesenchymal stem cells regulate ovarian aging in macaques

Tian Chuan1, 2, Zhu Xiangqing2, Yang Zailing1, 2, Yan Donghai2, Li Ye2, Wang Yanying2, Yang Yukun2, He Jie2, Lü Guanke2, Cai Xuemin2, Shu Liping1, He Zhixu3, Pan Xinghua1, 2   

  1. 1Guizhou Medical University, Tissue Engineering and Stem Cell Experimental Center, Guiyang 550004, Guizhou Province, China; 2The Basic Medical Laboratory of 920th Hospital of Joint Logistics Support Force of PLA, The Transfer Medicine Key Laboratory of Cell Therapy Technology of Yunan Province, The Integrated Engineering Laboratory of Cell Biological Medicine of State and Regions, Kunming 650032, Yunnan Province, China; 3Department of Pediatrics, Affiliated Hospital of Zunyi Medical University, Zunyi 563100, Guizhou Province, China
  • Received:2020-09-30 Revised:2020-10-12 Accepted:2020-11-13 Online:2022-03-08 Published:2021-10-28
  • Contact: Pan Xinghua, MD, Chief physician, Guizhou Medical University, Tissue Engineering and Stem Cell Experimental Center, Guiyang 550004, Guizhou Province, China; The Basic Medical Laboratory of 920th Hospital of Joint Logistics Support Force of PLA, The Transfer Medicine Key Laboratory of Cell Therapy Technology of Yunan Province, The Integrated Engineering Laboratory of Cell Biological Medicine of State and Regions, Kunming 650032, Yunnan Province, China
  • About author:Tian Chuan, Master candidate, Guizhou Medical University, Tissue Engineering and Stem Cell Experimental Center, Guiyang 550004, Guizhou Province, China; The Basic Medical Laboratory of 920th Hospital of Joint Logistics Support Force of PLA, The Transfer Medicine Key Laboratory of Cell Therapy Technology of Yunan Province, The Integrated Engineering Laboratory of Cell Biological Medicine of State and Regions, Kunming 650032, Yunnan Province, China
  • Supported by:
    the Major Science and Technology Project of Yunnan Science and Technology Plan Project, No. 2018ZF007 (to PXH); the Special Project of “Application-Review System”, No. 2019YGCO2 (to ZXQ)

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摘要：

文题释义：
骨髓间充质干细胞：作为再生医学领域中重要的种子细胞，具有的生物学特性主要包括：①自我更新能力；②多向分化潜能，可跨胚层分化；③抗氧化应激、抗炎症以及良好的免疫调节功能；④分泌外泌体和多种细胞因子。骨髓间充质干细胞的生物学特性使其有望成为治疗卵巢衰老的新“资源”。
卵巢衰老：是一个逐渐累积、多因素互作的复杂生物学过程，由卵泡数量减少和质量下降共同决定，其中基因和内分泌激素是影响卵泡数量的重要原因，而卵泡质量则与抗氧化系统、mtDNA突变以及端粒缩短等因素有关。
背景：卵巢作为女性生殖系统的核心器官，通过排卵与分泌激素行使女性生殖功能并影响全身的组织器官，随着人口老龄化加速以及多种诱因导致卵巢衰老人群不断增加，但卵巢衰老在整体上还处于初步研究阶段，依旧是尚待解决的科学问题。
目的：探索骨髓间充质干细胞对老年猕猴卵巢衰老的修复作用。
方法：健康老年雌猴10只，年龄23-27岁，体质量4.5-8.0 kg，随机分为老年对照组(n=4)，老年治疗组(n=6)。第4代幼年猕猴骨髓间充质干细胞经股静脉输注到老年猕猴体内，1次/d，隔日输注，连续输注3次；老年对照组猕猴同一时间输注等体积生理盐水。在输注细胞的第 8个月，安乐死处理猕猴取卵巢组织，称质量，40 g/L多聚甲醛固定，苏木精-伊红染色观察各级卵泡和卵巢结构变化；Tunel染色统计细胞凋亡率；Masson染色分析胶原蛋白所占面积比；免疫组织化学法观察CD34标记阳性血管数量。
结果与结论：①老年对照组卵巢脏器指数为1.8×10-5，老年治疗组卵巢脏器指数为6.0×10-5，两组比较差异有显著性意义(P < 0.05)；②苏木精-伊红染色结果显示老年治疗组可见原始、初级、次级、闭锁卵泡，未见成熟卵泡，髓质与间质明显，有少量钙结节；老年对照组基本无卵泡结构，局部只见闭锁卵泡，有大量脂肪组织填充；③Masson 染色结果显示老年治疗组卵巢组织胶原纤维所占面积比明显低于老年对照组，两组比较差异有显著性意义(P < 0.05)；④Tunel染色结果显示老年治疗组细胞凋亡率明显低于老年对照组，两组比较差异有显著性意义(P < 0.05)；⑤免疫组织化学结果显示老年治疗组血管数明显多于老年对照组，两组比较差异有显著性意义(P < 0.05)。⑥结果表明，骨髓间充质干细胞能够促进卵泡再生、改善卵巢组织结构、减少卵巢细胞凋亡、抑制卵巢纤维化进展、促进血管再生，以延缓甚至逆转卵巢衰老。

https://orcid.org/0000-0002-7954-8911(潘兴华) 

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程


关键词: 干细胞, 骨髓间充质干细胞, 卵巢衰老, 卵泡, 再生, 血管内皮细胞, 猕猴

Abstract: BACKGROUND: As the core organ of female reproductive system, ovary exerts female reproductive function through ovulation and hormone secretion, and affects the tissues and organs of the whole body. With the acceleration of population aging and a variety of inducements, the number of people with ovarian aging is increasing, but ovarian aging is still in the preliminary research stage, which is still a scientific problem to be solved.
OBJECTIVE: To investigate the effect of bone marrow mesenchymal stem cells on the repair of ovary of aging macaques.
METHODS:  Ten healthy old female monkeys, aged 23-27 years old and weighing 4.5-8.0 kg, were randomly divided into elderly control group (n=4) and elderly treatment group (n=6). The P4 bone marrow mesenchymal stem cells of young macaques were intravenously infused into elderly macaques, once a day, every other day, for three consecutive times. The elderly control group was infused with equal volume of normal saline at the same time. At the 8th month after the infusion of bone marrow mesenchyme stem cells, macaques were euthanized to take ovarian tissues, weighed, and fixed with 40 g/L paraformaldehyde. Hematoxylin-eosin staining was used to observe the structural changes of various levels of follicles for ovary. TUNEL staining was used to calculate the apoptosis rate. Masson staining was applied to analyze the area ratio of collagen. Immunohistochemistry was employed to observe the number of CD34-labeled positive blood vessel.
RESULTS AND CONCLUSION: (1) The ovarian organ index of the elderly control group was 1.8×10-5, and the ovarian organ index of the elderly treatment group was 6.0×10-5. There was significant difference between the two groups (P < 0.05). (2) Hematoxylin-eosin staining results showed that original, primary, secondary and atresic follicles were seen in the elderly treatment group, but mature follicle was not found; the medulla and stroma were obvious, and there were a few calcium nodules; the elderly control group basically had no follicular structure, only atresic follicle was seen in the local area, which was filled with a lot of fatty tissue. (3) Masson staining results showed that the area ratio of collagen fibers was significantly lower in the elderly treatment group than that in the elderly control group, and significant difference was found between the two groups (P < 0.05). (4) TUNEL staining results showed that the apoptosis rate was lower in the elderly treatment group than that in the elderly control group, and significant difference was found between the two groups (P < 0.05). (5) The results of immunohistochemistry showed that the number of blood vessels in the elderly treatment group was more than that in the elderly control group, and significant difference was found between the two groups (P < 0.05). (6) Results suggest that bone marrow mesenchymal stem cells can promote follicle regeneration, improve ovarian tissue structure, reduce ovarian cell apoptosis, inhibit the progression of ovarian fibrosis, and promote blood vessel regeneration, to delay even reverse ovarian senescence.

Key words: stem cells, bone marrow mesenchymal stem cells, ovarian aging, follicles, regeneration, vascular endothelial cell, macaque

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