承载GDNF修饰的脂肪间充质干细胞自体血小板凝胶对电烧伤后外周神经修复的作用机制研究

Peripheral nerve injury caused by electrical burns is one of serious problems demanding prompt solution during burn treatment. Studies have shown that stem cells and neurotrophic factors have important effects on peripheral nerve regeneration. Our previous study found that rat adipose-derived stem cells (ADSCs) transfected with glial cell line-derived neurotrophic factor (GDNF) helped to promote the functional recovery of rat sciatic nerve injury caused by electrical burns. However, disadvantages of local appliation of GDNF-ADSCs, namely easy to lose and to undergo apoptosis, greatly impede its exertion of sustained biological effects. Autologous platelet-rich gel (APG) features a good adhesive property that makes it easy to attach to the injured part when applied locally. Research has shown that APG can promote the growth and proliferation of ADSCs, as well as release GDNF which facilitates the regeneration of peripheral nerve injury. Therefore, in this project, we will firstly load GDNF-ADSCs onto APG for the preparation of GDNF-ADSCs-APG complex, and then observe the proliferation, differentiation and GDNF secretion of GDNF-ADSCs in GDNF-ADSCs-APG in vitro; Secondly, we will inject this complex to electrically injured sciatic nerve and observe the situation of axonal regeneration functional recovery of limbs. This project aims to explore the feasibility of GDNF-ADSCs-APG treatment of electrical injury to nerves, as well as elucidate the underlying mechanisms.

电烧伤造成的周围神经损伤是烧伤治疗中亟待解决的重要问题之一。研究表明干细胞与神经营养因子对外周神经损伤修复均具有重要作用。我们前期研究发现转染胶质细胞源性神经生长因子（GDNF）的大鼠脂肪干细胞（ADSCs）可以促进大鼠电烧伤坐骨神经功能的恢复。然而，由于局部应用GDNF-ADSCs易于流失及凋亡，导致其无法持续发挥有效的生物学作用。自体富血小板凝胶（APG）粘附性好，局部使用可附着于损伤部位。研究表明APG可促使ADSCs生长、增殖，同时自体可释放NF促进外周神经损伤修复。因此本课题拟将GDNF-ADSCs承载至APG,体外观察GDNF-ADSCs在APG中的增殖、分化及GDNF的分泌情况,体内将其注射于电烧伤坐骨神经，观察伤后神经轴突生长及肢体功能恢复情况，以探索GDNF-ADSCs-APG治疗神经电烧伤的可行性，并探讨其可能机制。

脂肪来源干细胞（ADSCs）适合于各种组织修复和伤口愈合。自体富血小板凝胶（APG）具有加速止血、封闭创面的特点，而且富含多种生长因子，其临床应用已越来越被医生认知。本研究的目的是探讨GDNF-ADSCs 承载至APG,体外观察GDNF-ADSCs 在APG 中的增殖、分化及GDNF 的分泌情况,体内将其注射于电烧伤坐骨神经，观察伤后神经轴突生长及肢体功能恢复情况。使用GDNF-ADSC与APG梯度浓度混合，并用EDU染色和免疫荧光检查体外细胞增殖和分化。通过将坐骨神经暴露于220V的电流3秒来构建大鼠坐骨神经电损伤模型。随后评估大鼠后肢运动功能和坐骨神经再生。ADSCs表面抗原CD90和CD105的高表达，CD34和CD45的低表达。我们发现在Lenti-GDNF感染后，ADSC中的GDNF蛋白表达升高。在GDNF-ADSCs-APG培养物中，随着培养时间的增加， ADSC增殖增强，神经元细胞核（GFAP）和胶质纤维酸性蛋白表达（GAP43）在GDNF-ADSCs-APG中上调。TGF-β表达逐渐降低，GDNF表达逐渐增高。此外，GDNF-ADSCs-APG处理后肢体运动功能和神经轴突生长得到改善。总之，我们的研究证明了ADSCs和APG在周围神经再生中的综合作用，并可能导致对电损伤患者有益的治疗。