Nerve growth factor promotes chondrogenic differentiation and inhibits hypertrophic differentiation of rabbit bone marrow mesenchymal stem cells

doi:10.12307/2025.005

Abstract

Abstract: BACKGROUND: Nerve growth factor is a protein that induces nerve growth and regulates biological behaviors such as proliferation and differentiation of mesenchymal stem cells.
OBJECTIVE: To investigate the promoting effect of nerve growth factor on chondrogenic differentiation of bone marrow mesenchymal stem cells.
METHODS: Rabbit bone marrow mesenchymal stem cells were isolated and cultured, and nerve growth factor was transfected into bone marrow mesenchymal stem cells by lentiviral transfection. The effects of nerve growth factor on the proliferation, migration, hypertrophic differentiation, and chondrogenic differentiation of bone marrow mesenchymal stem cells were detected by CCK-8 assay, cell scratch assay, alizarin red staining, and western blot assay, using the transfected null-loaded virus as control. To further investigate the promoting effect of nerve growth factor on the chondrogenic differentiation of bone marrow mesenchymal stem cells, interleukin 1β was added in bone marrow mesenchymal stem cells transfected with empty virus and nerve growth factor for 14 days. The expression of proteins related to chondrogenic differentiation and hypertrophic differentiation was detected by western blot assay.
RESULTS AND CONCLUSION: (1) CCK-8 assay results showed that nerve growth factor had no significant effect on the proliferation of bone marrow mesenchymal stem cells. (2) Compared with the control group, overexpression of nerve growth factor enhanced the migration ability of the cells, and the expression of cartilage-associated proteins type II collagen and SOX9 was up-regulated (P < 0.05), while the expression of hypertrophic-associated proteins type X collagen and Runx2 was down-regulated (P < 0.05). (3) Compared with the empty virus+interleukin 1β group, the expression of cartilage-associated proteins type II collagen and Sox9 was up-regulated (P < 0.05), and the expression of hypertrophy-associated proteins type X collagen and Runx2 was down-regulated after overexpression of nerve growth factor (P < 0.05). (4) The results indicated that nerve growth factor could promote the chondrogenic differentiation of bone marrow mesenchymal stem cells.

Key words: nerve growth factor, bone marrow mesenchymal stem cell, chondrogenic differentiation, osteoarthritis, chondrocyte, hypertrophic differentiation

CLC Number:

Yang Zhihang, Sun Zuyan, Huang Wenliang, Wan Yu, Chen Shida, Deng Jiang. Nerve growth factor promotes chondrogenic differentiation and inhibits hypertrophic differentiation of rabbit bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(7): 1336-1342.

Figures/Tables 2

2.1 兔骨髓间充质干细胞的形态首次换液后可见细胞呈集落样生长，继续培养3 d后可见细胞明显增多，细胞数量越加密集，呈纺锤形或梭形，培养至第7天后细胞继续增多，更加密集生长。经过传代处理后，细胞基本长满，见图1。 2.2 兔骨髓间充质干细胞多向分化能力鉴定及免疫荧光鉴定细胞表型诱导骨髓间充质干细胞向成骨、成软骨、成脂分化，采用茜素红、阿利新蓝、油红O染色鉴定骨髓间充质干细胞的多向分化能力，茜素红染色可见红色钙结节；阿利新蓝染色可见许多蓝色的颗粒，细胞质染成红色，聚集在一起，形成细胞外基质；油红O染色可见大量的褐色沉着，见图2。免疫荧光染色显示：骨髓间充质干细胞高表达CD29(+)、CD44(+)、CD90(+)，不表达CD34(-)、HLA-DR(-)，见图3。 2.3 NGF慢病毒转染构建稳定细胞株通过Western blot检测验证转染效率，与NC-NGF组相比，oe-NGF组NGF表达上调(P < 0.05)，见图4。 2.4 过表达NGF对骨髓间充质干细胞增殖活性的影响两组骨髓间充质干细胞增殖活性均显著升高，且oe-NGF组与NC-NGF组相比无显著差异，说明NGF对骨髓间充质干细胞的增殖活性无显著影响，见图5。 2.5 过表达NGF对骨髓间充质干细胞迁移能力的影响细胞划痕实验显示：培养24 h时两组骨髓间充质干细胞较0 h发生迁移，但并无明显区别；培养48 h时oe-NGF组无细胞区域基本被迁移的细胞填满，而NC-NGF组虽较24 h迁移的细胞增多，但较oe-NGF组无细胞区域较大，说明NGF可以促进骨髓间充质干细胞的迁移，见图6。 2.6 过表达NGF对骨髓间充质干细胞成骨及成软骨相关蛋白表达的影响 Western blot检测成软骨分化相关蛋白Ⅱ型胶原、SOX9和肥大分化相关蛋白Ⅹ型胶原、RUNX2的表达，与NC-NGF组相比，oe-NGF组成软骨相关蛋白Ⅱ型胶原、SOX9表达上调(P < 0.05)，肥大分化相关蛋白Ⅹ型胶原、RUNX2表达下调(P < 0.05)，见图7。 2.7 过表达NGF减弱白细胞介素1β对骨髓间充质干细胞软骨表型稳定的影响 Western blot检测成软骨分化相关蛋白Ⅱ型胶原、Sox9和肥大分化相关蛋白Ⅹ型胶原、Runx2的表达，与NC-NGF组相比，NC-NGF+白细胞介素1β组成软骨相关蛋白Ⅱ型胶原、SOX9表达下调(P < 0.05)，肥大分化相关蛋白Ⅹ型胶原、RUNX2表达上调(P < 0.05)；与NC-NGF+白细胞介素1β组相比，oe-NGF+白细胞介素1β组成软骨相关蛋白Ⅱ型胶原、SOX9表达上调(P < 0.05)，肥大相关蛋白Ⅹ型胶原、RUNX2表达下调(P < 0.05)，见图8。 2.8 过表达NGF减弱白细胞介素1β引起骨髓间充质干细胞的肥大分化与NC-NGF组相比，oe-NGF组骨髓间充质干细胞的钙含量降低(P < 0.05)，表明NGF可以抑制骨髓间充质干细胞的肥大分化；与NC-NGF组相比，NC-NGF+白细胞介素1β组骨髓间充质干细胞的钙含量升高(P < 0.05)；与NC-NGF+白细胞介素1β组相比，oe-NGF+白细胞介素1β组骨髓间充质干细胞的钙含量下降(P < 0.05)，表明NGF可以减弱白细胞介素1β引起骨髓间充质干细胞的肥大分化，见图9。"

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