切换至 "中华医学电子期刊资源库"
高级检索
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
全文 ( ) 下载PDF ( ) 导出引用
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]. 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]. 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
1
Tachibana M, Kuno T, Yaegashi N. Mitochondrial replacement therapy and assisted reproductive technology: a paradigm shift toward treatment of genetic diseases in gametes or in early embryos[J]. Reprod Med Biol, 2018, 17(4):421-433.
2
Kristensen SG, Pors SE, Andersen CY. Improving oocyte quality by transfer of autologous mitochondria from fully grown oocytes [J]. Hum Reprod, 2017, 32(4):725-732.
3
Tesarik J, Galán-Lázaro M, Mendoza-Tesarik R. Ovarian aging: molecular mechanisms and medical management[J]. Int J Mol Sci, 2021, 22(3):1371.
4
潘兴华. 脐带间充质干细胞治疗衰老退变的研究[J/OL]. 中华细胞与干细胞杂志（电子版）, 2020, 10(4):228.
5
Wang L, Mei Q, Xie Q, et al. A comparative study of mesenchymal stem cells transplantation approach to antagonize age-associated ovarian hypofunction with consideration of safety and efficiency[J]. J Adv Res, 2021, 38:245-259.
6
Salvatore G, De Felici M, Dolci S, et al. Human adipose-derived stromal cells transplantation prolongs reproductive lifespan on mouse models of mild and severe premature ovarian insufficiency[J]. Stem Cell Res Ther, 2021, 12(1):537.
7
Liu H, Jiang C, La B, et al. Human amnion-derived mesenchymal stem cells improved the reproductive function of age-related diminished ovarian reserve in mice through Ampk/FoxO3a signaling pathway[J]. Stem Cell Res Ther, 2021, 12(1):317.
8
Pan XH, Zhang XJ, Yao X, et al. Effects and mechanisms of mUCMSCs on ovarian structure and function in naturally ageing C57 mice[J]. J Ovarian Res, 2021, 14(1):133.
9
莫嘉辉,李欣禹,陈志宏等. 干细胞移植治疗睾丸衰老的现状与思考[J]. 器官移植, 2023, 14 (3): 319-326.
10
Yang YK, Li Y, Wang YY, et al. The effects of BMMSC treatment on lung tissue degeneration in elderly macaques[J]. Stem Cell Res Ther, 2021, 12(1):156.
11
Wang YY, Chang L, Zhu GH, et al. Mechanism of thymus rejuvenation in elderly macaques[J]. Rejuvenation Res, 2022, 25(5):223-232.
12
Tian C, He J, An YY, et al. Bone marrow mesenchymal stem cells derived from juvenile macaques reversed ovarian ageing in elderly macaques[J]. Stem Cell Res Ther, 2021, 12(1):460.
13
Long H, Wang Y, Wang L, et al. Age-related nomograms of serum anti-Mullerian hormone levels in female monkeys:comparison of rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) monkeys[J]. Gen Comp Endocrinol, 2018, 269:171-176.
14
Nichols SM, Bavister BD, Brenner CA, et al. Ovarian senescence in the rhesus monkey (Macaca mulatta)[J]. Hum Reprod, 2005, 20(1):79-83.
15
Colman RJ. Non-human primates as a model for aging[J]. Biochim Biophys Acta Mol Basis Dis, 2018, 1864(9 Pt A):2733-2741.
16
Kevenaar ME, Meerasahib MF, Kramer P, et al. Serum anti-mullerian hormone levels reflect the size of the primordial follicle pool in mice[J]. Endocrinology, 2006, 147(7):3228-3234.
17
Hansen KR, Hodnett GM, Knowlton N, et al. Correlation of ovarian reserve tests with histologically determined primordial follicle number[J]. Fertil Steril, 2011, 95(1):170-175.
18
Liu Q, Zhang J, Tang Y, et al. The effects of human umbilical cord mesenchymal stem cell transplantation on female fertility restoration in mice[J]. Curr Gene Ther, 2022, 22(4):319-330.
19
Xie Q, Xiong XL, Xiao N, et al. Mesenchymal stem cells alleviate DHEA-induced polycystic ovary syndrome (PCOS) by inhibiting inflammation in mice[J]. Stem Cells Int, 2019, 2019:9782373.
20
Di Micco R, Krizhanovsky V, Baker D, et al. Cellular senescence in ageing: from mechanisms to therapeutic opportunities[J]. Nat Rev Mol Cell Biol, 2021, 22(2):75-95.
21
Konala VBR, Bhonde R, Pal R. Secretome studies of mesenchymal stromal cells (MSCs) isolated from three tissue sources reveal subtle differences in potency[J]. In Vitro Cell Dev Biol Anim, 2020, 56(9):689-700.
22
Shin S, Lee J, Kwon Y, et al. comparative proteomic analysis of the mesenchymal stem cells secretome from adipose, bone marrow, placenta and wharton's jelly[J]. Int J Mol Sci, 2021, 22(2):845.
23
Lv FJ, Tuan RS, Cheung KM, et al. Concise review: the surface markers and identity of human mesenchymal stem cells[J]. Stem Cells, 2014, 32(6):1408-1419.
24
He H, Nagamura-Inoue T, Tsunoda H, et al. Stage-specific embryonic antigen 4 in Wharton's jelly-derived mesenchymal stem cells is not a marker for proliferation and multipotency[J]. Tissue Eng Part A, 2014, 20(7-8):1314-1324.
25
Kuroda Y, Kitada M, Wakao S, et al. Unique multipotent cells in adult human mesenchymal cell populations[J]. Proc Natl Acad Sci U S A, 2010, 107(19):8639-8643.
26
Kushida Y, Wakao S, Dezawa M. Muse cells are endogenous reparative stem cells[J]. Adv Exp Med Biol, 2018, 1103:43-68.
[1] 庄蕙嘉, 岳志成, 钟坤岑, 朱慧莉. 乳腺癌患者生育力保存的研究进展[J]. 中华乳腺病杂志(电子版), 2023, 17(04): 238-242.
[2] 林昌盛, 战军, 肖雪. 上皮性卵巢癌患者诊疗中基因检测及分子靶向药物治疗[J]. 中华妇幼临床医学杂志(电子版), 2023, 19(05): 505-510.
[3] 周东杰, 蒋敏, 范海瑞, 高玲玲, 孔祥, 卢丹, 王丽萍. 非编码RNA在卵泡发育成熟中作用及其机制的研究现状[J]. 中华妇幼临床医学杂志(电子版), 2023, 19(04): 387-393.
[4] 陈荟竹, 郭应坤, 汪昕蓉, 宁刚, 陈锡建. 上皮性卵巢癌"二元论模型"的分子生物学研究现状[J]. 中华妇幼临床医学杂志(电子版), 2023, 19(04): 394-402.
[5] 刘星辰, 刘娟, 魏宝宝, 刘洁, 刘辉. XIAP与XAF1异常表达与卵巢癌的相关性分析[J]. 中华妇幼临床医学杂志(电子版), 2023, 19(04): 419-427.
[6] 韩李念, 王君. 放射性皮肤损伤治疗的研究进展[J]. 中华损伤与修复杂志(电子版), 2023, 18(06): 533-537.
[7] 李晓晖, 上官昌盛, 向英, 裴芝皆, 车俊志, 谢飞. 3D腹腔镜袖状胃切除术后机体能量代谢与多囊卵巢综合征患者性激素水平关系[J]. 中华普外科手术学杂志(电子版), 2023, 17(05): 538-541.
[8] 唐英俊, 李华娟, 王赛妮, 徐旺, 刘峰, 李羲, 郝新宝, 黄华萍. 人脐带间充质干细胞治疗COPD小鼠及机制分析[J]. 中华肺部疾病杂志(电子版), 2023, 16(04): 476-480.
[9] 龙慧玲, 林蜜, 邵婷. 三维球体间充质干细胞培养技术的研究进展及其应用[J]. 中华细胞与干细胞杂志(电子版), 2023, 13(04): 229-234.
[10] 刘文慧, 吴涛, 张曦. 间充质干细胞联合血小板生成素受体激动剂在异基因造血干细胞移植后血小板恢复中的研究进展[J]. 中华细胞与干细胞杂志(电子版), 2023, 13(04): 242-246.
[11] 王红敏, 谢云波, 王彦虎, 王福生. 间充质干细胞治疗新冠病毒感染的临床研究进展[J]. 中华细胞与干细胞杂志(电子版), 2023, 13(04): 247-256.
[12] 杨蕴钊, 周诚, 石美涵, 赵静, 白雪源. 人羊水间充质干细胞对膜性肾病大鼠的治疗作用[J]. 中华肾病研究电子杂志, 2023, 12(04): 181-186.
[13] 宋艳琪, 任雪景, 王文娟, 韩秋霞, 续玥, 庄凯婷, 肖拓, 蔡广研. 间充质干细胞对顺铂诱导的小鼠急性肾损伤中细胞铁死亡的作用[J]. 中华肾病研究电子杂志, 2023, 12(04): 187-193.
[14] 梁宇同, 丁旭, 马国慧, 黄艳红. 间充质干细胞在宫腔粘连治疗中的研究进展[J]. 中华临床医师杂志(电子版), 2023, 17(05): 596-599.
[15] 于乾雪, 廖学梅, 孙龙龙, 范梦莹, 蒋明超, 孟慧, 李瑞基. 线粒体功能障碍与卵巢早衰的研究进展[J]. 中华诊断学电子杂志, 2023, 11(04): 283-288.
阅读次数
全文


摘要

版权所有 中华医学会 中华医学电子音像出版社有限责任公司  京ICP备14006079号-1  网络出版服务许可证：(署)网出证(京)字第075号