Introduction

Basic fibroblast growth factor (bFGF) is a promising cytokine in regenerative therapy for spinal cord injury. In this study, recombinant canine bFGF (rc-bFGF) was synthesized for clinical use in dogs, and the ability of rc-bFGF to differentiate canine bone marrow mesenchymal stem cells (BMSCs) into functional neurons was investigated.

Methods

The rc-bFGF was synthesized using a wheat germ cell-free protein synthesis system. The expression of rc-bFGF mRNA in the purification process was confirmed using a reverse transcription-polymerase chain reaction (RT-PCR). Western blotting was performed to confirm the antigenic property of the purified protein. To verify function of the purified protein, phosphorylation of extracellular signal-regulated kinase (ERK) was examined by in vitro assay using HEK293 cells. To compare the neuronal differentiation capacity of canine BMSCs in response to treatment with rc-bFGF, the cells were divided into the following four groups: control, undifferentiated, rh-bFGF, and rc-bFGF groups. After neuronal induction, the percentage of cells that had changed to a neuron-like morphology and the mRNA expression of neuronal markers were evaluated. Furthermore, to assess the function of the canine BMSCs after neuronal induction, changes in the intracellular Ca²⁺ concentrations after stimulation with KCl and l-glutamate were examined.

Results

The protein synthesized in this study was rc-bFGF and functioned as bFGF, from the results of RT-PCR, western blotting, and the expression of pERK in HEK293 cells. Canine BMSCs acquired a neuron-like morphology and expressed mRNAs of neuronal markers after neuronal induction in the rh-bFGF and the rc-bFGF groups. These results were more marked in the rc-bFGF group than in the other groups. Furthermore, an increase in intracellular Ca²⁺ concentrations was observed after the stimulation of KCl and l-glutamate in the rc-bFGF group, same as in the rh-bFGF group.

Conclusions

A functional rc-bFGF was successfully synthesized, and rc-bFGF induced the differentiation of canine BMSCs into voltage- and glutamate-responsive neuron-like cells. Our purified rc-bFGF may contribute, on its own, or in combination with canine BMSCs, to regenerative therapy for spinal cord injury in dogs.

中文翻译：

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重组犬碱性成纤维细胞生长因子诱导犬骨髓间充质干细胞分化为电压和谷氨酸反应性神经元样细胞

介绍

碱性成纤维细胞生长因子（bFGF）是脊髓损伤再生治疗中一种很有前途的细胞因子。本研究合成了用于犬临床的重组犬 bFGF (rc-bFGF)，并研究了 rc-bFGF 将犬骨髓间充质干细胞 (BMSCs) 分化为功能性神经元的能力。

方法

使用小麦生殖细胞无蛋白质合成系统合成 rc-bFGF。使用逆转录聚合酶链反应 (RT-PCR) 确认纯化过程中 rc-bFGF mRNA 的表达。进行蛋白质印迹以确认纯化蛋白质的抗原性。为了验证纯化蛋白的功能，通过体外检测细胞外信号调节激酶 (ERK) 的磷酸化使用 HEK293 细胞进行测定。为了比较犬骨髓间充质干细胞响应 rc-bFGF 处理的神经元分化能力，将细胞分为以下四组：对照组、未分化组、rh-bFGF 组和 rc-bFGF 组。神经元诱导后，评估已变为神经元样形态的细胞百分比和神经元标志物的 mRNA 表达。此外，为了评估神经元诱导后犬骨髓间充质干细胞的功能，检查了用 KCl 和 L-谷氨酸刺激后细胞内 Ca 2+ 浓度的^变化。

结果

从 RT-PCR、蛋白质印迹和 HEK293 细胞中 pERK 表达的结果来看，本研究中合成的蛋白质是 rc-bFGF，并作为 bFGF 发挥作用。犬骨髓间充质干细胞在 rh-bFGF 和 rc-bFGF 组中获得神经元样形态并在神经元诱导后表达神经元标志物的 mRNA。这些结果在 rc-bFGF 组中比在其他组中更显着。此外，在 rc-bFGF 组中，在 KCl 和l-谷氨酸刺激后，观察到细胞内 Ca ²⁺浓度的增加，这与 rh-bFGF 组相同。

结论

成功合成了功能性rc-bFGF，rc-bFGF诱导犬BMSCs分化为电压和谷氨酸反应性神经元样细胞。我们纯化的 rc-bFGF 可能单独或与犬骨髓间充质干细胞联合用于犬脊髓损伤的再生治疗。