中国组织工程研究 ›› 2025, Vol. 29 ›› Issue (36): 7709-7718.doi: 10.12307/2025.535

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

年轻大鼠骨髓间充质干细胞来源外泌体逆转老龄大鼠骨髓间充质干细胞衰老

张熊劲夫,陈奕达,程歆怡,刘岱珲,施  勤   

  1. 苏州大学附属第一医院骨科,苏州大学骨科研究所,江苏省苏州市   215006
  • 收稿日期:2024-04-15 接受日期:2024-06-21 出版日期:2025-12-28 发布日期:2025-03-01
  • 通讯作者: 施勤,博士,教授,苏州大学附属第一医院骨科,苏州大学骨科研究所,江苏省苏州市 215006
  • 作者简介:张熊劲夫,女,1998 年生,云南省昆明市人,纳西族,2021 年苏州大学毕业,主要从事骨质疏松研究。
  • 基金资助:
    国家自然科学基金面上项目(82172485),项目负责人:施勤

Exosomes derived from bone marrow mesenchymal stem cells of young rats to reverse senescence in aged rat bone marrow mesenchymal stem cells

Zhang Xiongjinfu, Chen Yida, Cheng Xinyi, Liu Daihui, Shi Qin   

  1. Department of Orthopedics, First Affiliated Hospital of Soochow University, Institute of Orthopedics of Soochow University, Suzhou 215006, Jiangsu Province, China
  • Received:2024-04-15 Accepted:2024-06-21 Online:2025-12-28 Published:2025-03-01
  • Contact: Shi Qin, MD, Professor, Department of Orthopedics, First Affiliated Hospital of Soochow University, Institute of Orthopedics of Soochow University, Suzhou 215006, Jiangsu Province, China
  • About author:Zhang Xiongjinfu, Department of Orthopedics, First Affiliated Hospital of Soochow University, Institute of Orthopedics of Soochow University, Suzhou 215006, Jiangsu Province, China
  • Supported by:
    National Natural Science Foundation of China, No. 82172485 (to SQ)

摘要:

文题释义:

细胞衰老:是指细胞在执行生命活动过程中,随着时间的推移,细胞增殖与分化能力和生理功能逐渐发生衰退的变化过程。
微小RNA:外泌体内包含多种来自分泌细胞的微小RNA,微小RNA与靶细胞基因组结合,影响靶细胞相关基因转录和蛋白表达。

摘要
背景:骨髓间充质干细胞是骨形成的主要效应细胞,随着年龄的增加,骨髓间充质干细胞的再生能力减弱、分化功能受损,导致骨质疏松。因此,恢复老龄骨髓间充质干细胞的再生能力和细胞功能对骨质疏松的有效治疗至关重要。
目的:探究年轻大鼠第3,11代骨髓间充质干细胞来源外泌体对老年大鼠骨髓间充质干细胞衰老的影响。
方法:分离培养6-8周龄雌性SD大鼠骨髓间充质干细胞,分别传代至第3代和第11代,随后提取第3,11代骨髓间充质干细胞来源外泌体。分离培养18月龄雌性SD大鼠骨髓间充质干细胞,传代至第3代,分为3组:对照组为常规培养,其他2组用第3,11代骨髓间充质干细胞来源外泌体干预。外泌体干预48 h后,利用β-半乳糖苷酶染色试剂盒检测细胞核内β-半乳糖苷酶的表达,qRT-PCR检测衰老相关基因的表达。通过Small RNA测序,比较第3,11代骨髓间充质干细胞来源外泌体内 miRNA 的表达差异。
结果与结论: ①与对照组和第11代骨髓间充质干细胞来源外泌体组相比,第3代骨髓间充质干细胞来源外泌体组老龄大鼠骨髓间充质干细胞的β-半乳糖苷酶活性显著降低;②与对照组相比,第3代骨髓间充质干细胞来源外泌体组衰老相关基因p21和p16的表达显著降低(P < 0.05),而第11代骨髓间充质干细胞来源外泌体组衰老相关基因p21和p16的表达无显著差异;③测序结果表明,两种外泌体内miRNA的表达存在显著差异,其中表达差异最显著的 miRNA是 let-7c-5p、let-7b-5p 、miR-320-3p 和 miR-26a-5p,KEGG分析结果显示显著性差异 miRNA富集通路包含mTOR、AMPK等衰老相关信号通路。上述结果表明,第3代骨髓间充质干细胞来源外泌体具有逆转老龄大鼠骨髓间充质干细胞衰老的能力。

关键词: 骨质疏松, 外泌体, 骨髓间充质干细胞, miRNA, 衰老, 工程化细胞外囊泡

Abstract: BACKGROUND: Bone marrow mesenchymal stem cells are the main effector cells for bone formation. With the increase of age, the regenerative ability of bone marrow mesenchymal stem cells is weakened and the differentiation function is impaired, leading to poor osteoporosis. Therefore, restoring the regenerative capacity and cellular function of aged bone marrow mesenchymal stem cells is essential for the effective treatment of osteoporosis.
OBJECTIVE: To investigate the effects of passage 3 and passage 11 bone marrow mesenchymal stem cells-derived exosomes of young rats on the aging of bone marrow mesenchymal stem cells derived from elderly rats.
METHODS: Bone marrow mesenchymal stem cells from 6-8-week-old female SD rats were isolated and cultured, and passaged to the passages 3 and 11, respectively. Then, exosomes from passages 3 and 11 bone marrow mesenchymal stem cells were extracted. Bone marrow mesenchymal stem cells from 18-month-old female SD rats were isolated and cultured, passaged to passage 3, and divided into 3 groups. The control group was routinely cultured, and the other two groups were intervened with exosomes from passages 3 and 11 bone marrow mesenchymal stem cells. After 48 hours of exosome intervention, the expression of β-galactosidase in the nucleus was detected by β-galactosidase staining kit. The expression of aging-related genes was detected by qRT-PCR. The expression differences of miRNA in exosomes from passages 3 and 11 bone marrow mesenchymal stem cells were compared by Small RNA sequencing.
RESULTS AND CONCLUSION: (1) Compared with the control group and passage 11 bone marrow mesenchymal stem cell-derived exosomes group, the β-galactosidase activity of bone marrow mesenchymal stem cells of aged rats was significantly lower in the passage 3 bone marrow mesenchymal stem cell-derived exosomes group. (2) Compared with the control group, the expression of aging-related genes p21 and p16 was significantly reduced in the passage 3 bone marrow mesenchymal stem cell-derived exosome group (P < 0.05), while there was no significant difference in the expression of aging-related genes p21 and p16 in the passage 11 bone marrow mesenchymal stem cell-derived exosome group. (3) Sequencing results showed that there was a significant difference in the expression of miRNAs in the two exosomes, among which the miRNAs with the most significant expression differences were let-7c-5p, let-7b-5p, miR-320-3p, and miR-26a-5p. KEGG analysis results showed that significantly different miRNA enrichment pathways include mTOR, AMPK and other aging-related signaling pathways. The above results indicate that passage 3 bone marrow mesenchymal stem cell-derived exosomes have the ability to reverse the aging of bone marrow mesenchymal stem cells in aged rats.

Key words: osteoporosis, exosomes, bone marrow mesenchymal stem cell, miRNA, aging, engineered extracellular vesicles

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