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

doi:10.12307/2025.535

Abstract

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

CLC Number:

Zhang Xiongjinfu, Chen Yida, Cheng Xinyi, Liu Daihui, Shi Qin . Exosomes derived from bone marrow mesenchymal stem cells of young rats to reverse senescence in aged rat bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(36): 7709-7718.

Figures/Tables 5

2.4 第3，11代骨髓间充质干细胞来源外泌体的粒径分布纳米颗粒分析结果显示，第3，11代骨髓间充质干细胞来源外泌体的粒径大小主要分布在150 nm 左右，检测结果呈现单峰，说明提取出的外泌体纯度较高，见图4。 2.5 第3，11代骨髓间充质干细胞来源外泌体的标志蛋白表达第3，11代骨髓间充质干细胞来源外泌体均表达CD63和TSG101，符合外泌体的表征验证，见图5。 2.6 老龄大鼠骨髓间充质干细胞对年轻大鼠第3，11代骨髓间充质干细胞来源外泌体的内化倒置荧光显微镜观察结果显示，年轻大鼠第3，11代骨髓间充质干细胞来源外泌体均能被老龄大鼠骨髓间充质干细胞摄取，二者内化情况一致，无明显差别，外泌体均聚集在老龄大鼠骨髓间充质干细胞的细胞质和细胞核周围，进一步表明老龄大鼠骨髓间充质干细胞能够内化年轻大鼠第3，11代骨髓间充质干细胞来源外泌体，被摄取的外泌体能够进一步进入细胞核内发挥生物学作用，见图6。 2.7 第3代骨髓间充质干细胞来源外泌体促进老龄大鼠骨髓间充质干细胞的增殖老龄大鼠骨髓间充质干细胞接种培养1，3，5 d后，能够正常增殖，并且加入第3代骨髓间充质干细胞来源外泌体处理后老龄大鼠骨髓间充质干细胞的增殖速度增加，加入第11代骨髓间充质干细胞来源外泌体后老龄大鼠骨髓间充质干细胞的增殖速度下降。上述结果表明第3代骨髓间充质干细胞来源外泌体能够促进老龄大鼠骨髓间充质干细胞的增殖，见图7。 2.8 第3代骨髓间充质干细胞来源外泌体促进老龄大鼠骨髓间充质干细胞成骨分化相关基因的表达与第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Bmp2、Spp1和Runx2的mRNA表达均上调，见图8。 2.9 第3代骨髓间充质干细胞来源外泌体提高老龄大鼠骨髓间充质干细胞成骨分化后的碱性磷酸酶活性碱性磷酸酶染色结果表明，加入第3代骨髓间充质干细胞来源外泌体后，老龄大鼠骨髓间充质干细胞的碱性磷酸酶表达量显著增加，见图9。 2.10 第3代骨髓间充质干细胞来源外泌体降低老龄大鼠骨髓间充质干细胞衰老相关基因表达与第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p21的mRNA表达下调了约48%，p16的mRNA表达下调了约30%(P < 0.05)，见图10。 "

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