Ji Wenchen, Zhang Xiaowei, Ji Le, Wang Kunzheng, Qiu Yusheng
Department of Orthopedics, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
Department of Orthopedics, The Third Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710068, P.R. China.
Mol Med Rep. 2015 Oct;12(4):4981-8. doi: 10.3892/mmr.2015.4099. Epub 2015 Jul 20.
Tissue engineering is a promising method that may be used to treat spinal cord injury (SCI). The underlying repair mechanism of tissue engineering involves the stable secretion of neurotrophins from seed cells, which eventually differentiate into neurons; therefore, the selection of appropriate seed cells, which stably secrete neurotrophins that easily differentiate into neurons requires investigation. Adipose‑derived stem cells (ADSCs), which are adult SCs, are advantageous due to convenience sampling and easy expansion; therefore, ADSCs are currently the most popular type of seed cell. Brain‑derived neurotrophic factor (BDNF) and neurotrophin‑3 (NT‑3) possess superior properties, when compared with other neurotrophic factors, in the maintenance of neuronal survival and promotion of SC differentiation into neurons. The present study used two lentiviruses, which specifically express BDNF and NT‑3 [Lenti‑BDNF‑green fluorescent protein (GFP), Lenti‑NT‑3‑red fluorescent protein (RFP)], to transfect third‑generation ADSCs. Three types of seed cell were obtained: i) Seed cells overexpressing BDNF (ADSC/Lenti‑BDNF‑GFP); ii) seed cells overexpressing NT‑3 (ADSC/Lenti‑NT‑3‑RFP); and iii) seed cells overexpressing BDNF and NT‑3 (ADSC/Lenti‑BDNF‑GFP and NT‑3‑RFP). The transfected cells were then induced to differentiate into neurons and were divided into a further four groups: i) The BDNF and NT‑3 co‑overexpression group; ii) the BDNF overexpression group; iii) the NT‑3 overexpression group; and iv) the control group, which consisted of untransfected ADSCs. The results of the present study demonstrate that BDNF and NT‑3 expression was higher 10 days after induction, as detected by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blotting. Neuron‑specific enolase is a neuronal marker, the expression of which was highest in the BDNF and NT‑3 co‑overexpression group, followed by the BDNF overexpression group and then by the NT‑3 overexpression group. The lowest expression levels of NSE were detected in the control group, as determined by RT‑qPCR, western blotting and immunofluorescent staining. These results indicate that BDNF and NT‑3 exert a synergistic effect, which may promote the neuronal differentiation of ADSCs. The present study provides a solid theoretical foundation for future experiments regarding the use of tissue engineering technology for the treatment of SCI.
组织工程是一种有前景的方法，可用于治疗脊髓损伤（SCI）。组织工程的潜在修复机制涉及种子细胞稳定分泌神经营养因子，这些因子最终分化为神经元；因此，需要研究选择能稳定分泌易于分化为神经元的神经营养因子的合适种子细胞。脂肪来源干细胞（ADSCs）作为成体干细胞，因取材方便且易于扩增而具有优势；因此，ADSCs是目前最常用的种子细胞类型。与其他神经营养因子相比，脑源性神经营养因子（BDNF）和神经营养因子-3（NT-3）在维持神经元存活和促进干细胞分化为神经元方面具有更优异的特性。本研究使用两种特异性表达BDNF和NT-3的慢病毒[慢病毒-BDNF-绿色荧光蛋白（GFP）、慢病毒-NT-3-红色荧光蛋白（RFP）]转染第三代ADSCs。获得了三种类型的种子细胞：i）过表达BDNF的种子细胞（ADSC/慢病毒-BDNF-GFP）；ii）过表达NT-3的种子细胞（ADSC/慢病毒-NT-3-RFP）；iii）过表达BDNF和NT-3的种子细胞（ADSC/慢病毒-BDNF-GFP和NT-3-RFP）。然后将转染后的细胞诱导分化为神经元，并进一步分为四组：i）BDNF和NT-3共过表达组；ii）BDNF过表达组；iii）NT-3过表达组；iv）对照组，由未转染的ADSCs组成。本研究结果表明，通过逆转录-定量聚合酶链反应（RT-qPCR）和蛋白质免疫印迹法检测，诱导10天后BDNF和NT-3的表达更高。神经元特异性烯醇化酶是一种神经元标志物，其表达在BDNF和NT-3共过表达组中最高，其次是BDNF过表达组，然后是NT-3过表达组。通过RT-qPCR、蛋白质免疫印迹法和免疫荧光染色测定，对照组中NSE的表达水平最低。这些结果表明，BDNF和NT-3发挥协同作用，可能促进ADSCs向神经元分化。本研究为未来利用组织工程技术治疗SCI的实验提供了坚实的理论基础。
