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中华细胞与干细胞杂志(电子版)

中华细胞与干细胞杂志(电子版) ›› 2023, Vol. 13 ›› Issue (04) : 210 -219. doi: 10.3877/cma.j.issn.2095-1221.2023.04.003

论著

高活性间充质干细胞干预猕猴卵巢衰老的研究
李晔, 何洁, 胡锦秀, 王金祥, 田川, 潘杭, 陈梦蝶, 赵晓娟, 叶丽, 张敏, 潘兴华†()   
  1. 650032　昆明，中国人民解放军联勤保障部队第九二〇医院基础医学实验室，干细胞与免疫细胞生物医药技术国家地方联合工程研究中心，云南省细胞治疗技术转化医学重点实验室，昆明市干细胞与再生医学重点实验室；650032　昆明，昆明医科大学第九二〇医院临床学院
    650106　昆明，奥辰生物（云南）有限公司
    650032　昆明，中国人民解放军联勤保障部队第九二〇医院基础医学实验室，干细胞与免疫细胞生物医药技术国家地方联合工程研究中心，云南省细胞治疗技术转化医学重点实验室，昆明市干细胞与再生医学重点实验室
    650051　昆明，云南维权司法鉴定中心
  • 收稿日期:2022-12-19 出版日期:2023-08-01
  • 通信作者: 潘兴华
  • 基金资助:
    全军实验动物专项(SYDW[2020]19); 云南省基础研究专项(202101AT070212); 联勤保障部队第九二〇医院科技计划项目(2020YGD05)

Highly active mesenchymal stem cells interfere with ovarian aging in macaques

Ye Li, Jie He, Jinxiu Hu, Jinxiang Wang, Chuan Tian, Hang Pan, Mengdie Chen, Xiaojuan Zhao, Li Ye, Min Zhang, Xinghua Pan†()   

  1. Basic Medical Laboratory, 920th Hospital of Joint Logistics Support Force, PLA, the Stem Cells and Immune Cells Biomedical Techniques Integrated Engineering Research Center of State and Regions, Cell Therapy Technology Transfer Medical Key Laboratory of Yunnan Province, Kunming Key Laboratory of Stem Cell and Regenerative Medicine, Kunming 650032, China; Clinical College of the 920th Hospital of Kunming Medical University, Kunming 650032, China
    Aochen Biotechnology (Yunnan) Co., Ltd., Kunming 650106, China
    Basic Medical Laboratory, 920th Hospital of Joint Logistics Support Force, PLA, the Stem Cells and Immune Cells Biomedical Techniques Integrated Engineering Research Center of State and Regions, Cell Therapy Technology Transfer Medical Key Laboratory of Yunnan Province, Kunming Key Laboratory of Stem Cell and Regenerative Medicine, Kunming 650032, China
    Yunnan Rights Protection Judicial Expertise Center, Kunming 650051, China
  • Received:2022-12-19 Published:2023-08-01
  • Corresponding author: Xinghua Pan
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引用本文：

李晔, 何洁, 胡锦秀, 王金祥, 田川, 潘杭, 陈梦蝶, 赵晓娟, 叶丽, 张敏, 潘兴华. 高活性间充质干细胞干预猕猴卵巢衰老的研究[J/OL]. 中华细胞与干细胞杂志(电子版), 2023, 13(04): 210-219.

Ye Li, Jie He, Jinxiu Hu, Jinxiang Wang, Chuan Tian, Hang Pan, Mengdie Chen, Xiaojuan Zhao, Li Ye, Min Zhang, Xinghua Pan. Highly active mesenchymal stem cells interfere with ovarian aging in macaques[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2023, 13(04): 210-219.

目的

研究一种高活性间充质干细胞（HA-MSCs）对衰老猕猴卵巢结构与功能的干预作用。

方法

分别使用TeSR-E8培养基和含10%胎牛血清的DMEM/F12培养基从青年猕猴骨髓中分离、扩增得到猕猴HA-MSCs与骨髓间充质干细胞（BMSCs），流式细胞仪检测细胞表面标志物，并进行成脂、成骨和成软骨诱导分化能力鉴定。然后将这2种细胞按1×107个细胞/kg的剂量，1日1次，连续3 d静脉输注给卵巢衰老猕猴，于治疗前1天、治疗后1、2、3、5个月检测外周血抗苗勒氏管激素（AMH）水平，治疗后第5个月将猕猴安乐死，HE染色观察卵巢的组织病理结构，Masson染色评价卵巢纤维化程度变化，免疫组织化学染色检测卵巢p16、p21衰老相关蛋白的表达水平。两组间比较采用t检验，多组间比较采用单因素方差分析，组间两两比较采用Tukey方法。

结果

HA-MSCs呈短小梭形，核质比高，形态均一。BMSCs体积较大，呈典型的成纤维细胞形态，有少量杂细胞。HA-MSCs的成脂、成骨和成软骨分化能力均强于BMSCs。与老年模型组比较，HA-MSCs治疗组在治疗后2个月内AMH水平[（1.85 ± 0.21）比（0.29 ± 0.15）ng/mL]增加，该组卵巢组织可见各级卵泡，纤维化程度降低（P均< 0.001）。与老年模型组比较，BMSCs治疗组在治疗后1个月时AMH水平[（1.26 ± 0.28）比（0.36 ± 0.16）ng/mL]增加，与HA-MSCs治疗组比较，BMSCs治疗组在治疗后2个月时AMH水平[（1.85 ± 0.21）比（0.60 ± 0.20）ng/mL]降低，该组可见少量未成熟卵泡及原始卵泡，纤维化程度降低（P均< 0.001）。老年模型组AMH水平最低，卵巢组织偶见闭锁卵泡，广泛纤维化。卵巢p16、p21蛋白表达水平在老年模型组最高，HA-MSCs治疗组最低，BMSCs治疗组次之。

结论

HA-MSCs提高衰老卵巢的储备能力，减轻卵巢纤维化，减少卵巢衰老相关蛋白的表达，重启卵泡生成功能，在治疗卵巢衰老上相较BMSCs效果更佳。

关键词: 间充质干细胞, 衰老, 猕猴, 卵巢
Objective

To study the intervention effect of highly active mesenchymal stem cells (HA-MSCs) on the structure and function of the ovary in aging macaques.

Methods

Macaque HA-MSCs and bone marrow mesenchymal stem cells (BMSCs) were isolated and expanded from young macaque bone marrow using TeSR-E8 medium and DMEM/F12 medium containing 10% fetal bovine serum, respectively. Cell surface markers were detected by flow cytometry, and adipogenic, osteogenic, and chondrogenic differentiation capacities were identified. These two kinds of cells were then infused intravenously into ovarian senescent macaques at a dose of 1×107 cells/kg, once a day for 3 consecutive days. The peripheral blood anti-Mullerian hormone (AMH) levels were measured before and 1, 2, 3 and 5 months after treatment, and the macaques were euthanized at the 5th month after treatment. The histopathological structure of the ovaries was observed by HE staining, and the degree of ovarian fibrosis alteration was evaluated by Masson staining. Immunohistochemical staining was employed to detect the expression levels of ageing-associated markers p16 and p21 in the ovaries.

Results

HA-MSCs are short and fusiform with a high nuclear/cytoplasmic ratio and uniform morphology. BMSCs are large in size, showing typical fibroblast morphology, with few a small number of miscellaneous cells. The adipogenic, osteogenic, and chondrogenic differentiation capacities of HA-MSCs are all superior to those of BMSCs. Compared with the aged model group, the HA-MSCs treatment group showed an increase in AMH levels [ (1.85 ± 0.21) vs (0.29 ± 0.15) ng/mL] within two months post-treatment, with ovarian tissues exhibiting follicles of various stages and a decreased degree of fibrosis (all P < 0.001) . Compared to the aged model group, the BMSCs treatment group demonstrated an increase in AMH levels [ (1.26 ± 0.28) vs (0.36 ± 0.16) ng/mL] one month post-treatment. When compared with the HA-MSCs treatment group, the BMSCs treatment group showed a decrease in AMH levels [ (1.85 ± 0.21) vs (0.60 ± 0.20) ng/mL] two months after treatment. The ovarian tissues of this group revealed a small number of immature and primordial follicles, along with a reduced dreduction in fibrosis (all P < 0.001) . The elderly model group had the lowest AMH levels, with occasional atretic follicles and extensive fibrosis in the ovarian tissues. The expression levels of p16 and p21 proteins in the ovaries were highest in the elderly model group, lowest in the HA-MSCs treatment group, and intermediate in the BMSCs treatment group.

Conclusion

HA-MSCs can increase the reserve capacity of the aging ovary, and significantly mitigate ovarian fibrosis and the expression levels of ovarian aging-related proteins, and restart the follicle formation function. HA-MSCs transplantation therapy can be a better intervention for the treatment of ovarian aging than BMSCs.

Key words: Mesenchymal stem cells, Aging, Macaques, Ovary
图1 倒置相差显微镜下观察猕猴的细胞形态注：a，c图为P3 HA-MSCs，体积较小，呈短小梭形，核质比高，边界清晰，形态均一，呈鱼群状排列；b，d图为P3 BMSCs，体积较大，呈典型的成纤维细胞形态，有少量杂细胞。a ~ b图×4，c ~ d图×10
图2 猕猴HA-MSCs表面标志物表达流式图
图3 猕猴BMSCs表面标志物表达流式图
表1 HA-MSCs和BMSCs表面标志物表达水平
细胞类型 CD73 CD90 CD105 CD34 CD45 SSEA3 SSEA4 TRA-1-60
HA-MSCs 99.40% 98.70% 98.80% 0.72% 0.31% 26.90% 19.60% 15.70%
BMSCs 96.80% 97.20% 97.50% 1.92% 0.71% 0 25.80% 0
图4 猕猴HA-MSCs和BMSCs三系分化能力比较注：a，d图为成脂分化（油红O染色，×40）；b，e图为成骨分化（茜素红染色，×10）；c，f图为成软骨分化（阿利新蓝染色，×4）；aP < 0.001
图5 猕猴血清AMH水平变化注：HA-MSCs与BMSCs治疗后每个时间点分别与老年模型组比较，aP < 0.01，bP < 0.001，ns为差异无统计学意义
图6 光学显微镜下观察卵巢组织（HE染色）注：a，d图为老年模型组；b，e图为HA-MSCs治疗组；c，f图为BMSCs治疗组；红色箭头为原始卵泡，绿色箭头为初级卵泡，蓝色箭头为次级卵泡，黑色箭头为闭锁卵泡；a ~ c图×10，d ~ f图×20
图7 光学显微镜下观察卵巢组织（Masson染色，×20）注：a图为老年模型组，b图为HA-MSCs治疗组，c图为BMSCs治疗组；黄色箭头为胶原纤维，黑色箭头为闭锁卵泡；d图为HA-MSCs治疗组，老年模型组及BMSCs治疗组卵巢组织胶原面积比；与老年模型组比较，aP < 0.01，与BMSCs治疗组比较，bP < 0.001
图8 光学显微镜下观察卵巢p16蛋白免疫组织化学染色（×20）注：a图为老年模型组，b图为HA-MSCs治疗组，c图为BMSCs治疗组；深棕色表示强阳性，棕黄色为中度阳性，浅黄色为弱阳性，蓝色为细胞核；d图为HA-MSCs治疗组，老年模型组及BMSCs治疗组卵巢蛋白水平比较；与老年模型组比较，aP < 0.01，与BMSCs治疗组比较，bP < 0.001
图9 光学显微镜下观察卵巢p21蛋白免疫组织化学染色（×20）注：a图为老年模型组，b图为HA-MSCs治疗组，c图为BMSCs治疗组；深棕色表示强阳性，棕黄色为中度阳性，浅黄色为弱阳性，蓝色为细胞核；d图为HA-MSCs治疗组，老年模型组及BMSCs治疗组卵巢蛋白水平比较；与老年模型组比较，aP < 0.01，与BMSCs治疗组比较，bP < 0.001，ns为差异无统计学意义
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