目的 探讨移植胶质细胞源性神经营养因子（glial cell line derived neurotrophic factor，GDNF）基因修饰的神经干细胞（neural stem cells，NSCs）对暂时性缺血性脑卒中大鼠的神经保护。 方法 用GDNF重组腺病毒载体转染新生大鼠NSCs（GDNF/NSCs），分化培养7 d后，行免疫细胞化学染色检测微管相关蛋白2（MAP2）。采用改良的插线法制作暂时性脑缺血再灌注模型，3 d后经脑室分别移植生理盐水、NSCs和GDNF/NSCs。于再灌注后1、2、3、5、7周末处死大鼠，行免疫组织化学染色观察移植细胞在脑内的神经元分化及星形胶质细胞在缺血区形成胶质界膜情况，行Luxol fast blue（LFB）染色显示神经纤维损伤情况。 结果 GDNF/NSCs体外分化为MAP2+细胞的比例显著高于NSCs的分化。移植细胞在脑内分化为MAP2+细胞，于再灌注第5周分化达高峰，GDNF/NSCs组于再灌注第3~7周，其MAP2+细胞显著高于NSCs组。各组缺血区由星形胶质细胞形成的血管胶质界膜存在不同程度的破坏，其连续性中断。对照组在各个时间点，血管胶质界膜损伤严重，完整性差，两细胞移植组，其胶质界膜随时间延长逐渐完整，GDNF/NSCs组早于NSCs组完善对胶质界膜的修复。此外，GDNF/NSCs组的神经纤维损伤修复优于NSCs组。 结论 GDNF/NSCs比NSCs对暂时性缺血性脑卒中大鼠模型有更好的神经保护作用，可能是与GDNF提高了NSCs在脑内的神经元分化，增强了NSCs对胶质界膜及神经纤维修复有关。
Abstract
Objective To investigate the neuroprotective effects of transplantation of glial cell-derived trophic factor (GDNF) gene-modified neural stem cells (NSCs) on temporary cerebral ischemia in rats. Methods Primary neonatal rat NSCs were transfected with GDNF plasmid (GDNF/NSCs). After 7 days they were induced to differentiate, immunohistochemistry staining was used to detect the expression of MAP2+ positive cells. Rats were subjected to two-hour middle cerebral artery occlusion and reperfusion, followed by infusion of saline, NSCs or GDNF/NSCs after reperfusion respectively. All rats were sacrificed at 1, 2, 3, 5, and 7 weeks after reperfusion. Immunohistochemistry staining was performed to identify the neuronal differentiation from implanted cells and to assess glia limitans perivascularis formed by astrocyte. Luxol fast blue（LFB）staining was used to observe nerve fiber damages. Results MAP2+ cells from GDNF/NSCs were higher than those from NSCs in vitro. Implanted cells differentiated into MAP2+ cells, which reached the peak at 5 weeks after reperfusion. MAP2+ cells from grafted GDNF/NSCs at 3~7 weeks after reperfusion were higher than those from grafted NSCs. The glia limitans perivascularis in ischemic areas encountered varying degrees of damages in all groups, and the damages in control group at different time points were serious, while the damages in two transplantation groups were gradually repaired over time, and the repair in GDNF/NSCs group was earlier than that in NSCs group. Furthermore, the recovery of nerve fiber in GDNF/NSCs group was stronger than that in NSCs group. Conclusions GDNF/NSCs provide better neuroprotective effect for temporary cerebral ischemia than NSCs, which may be related to GDNF enhancing neuronal differentiation and restoration on glia limitans and nerve fibers of NSCs.
关键词
神经干细胞 /
基因治疗 /
胶质细胞源性神经营养因子 /
脑卒中 /
胶质界膜
Key words
Neural stem cells; Gene therapy; Glial cell line derived neurotrophic factor; Stroke /
Glia limitans
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参考文献
[1] Gao XQ, Wu DD, Dou L, et al. Protective effects of mesenchymal stem cells overexpressing extracellular regulating kinase 1/2 against stroke in rats[J]. Brain Res Bull, 2019, 149: 42-52.
[2] Chen B, Gao XQ, Yang CX, et al. Neuroprotective effect of grafting GDNF gene-modified neural stem cells on cerebral ischemia in rats[J]. Brain Res, 2009, 1284: 1-11.
[3] Beck KD, Valverde J, Alexi T, et al. Mesencephalic dopaminergic neurons protected by GDNF from axotomyinduced degeneration in the adult brain[J]. Nature, 1995, 373(6512): 339-341.
[4] Kobayashi T, Ah1enius H, Thored P, et al. Intracere bralinfusion of glial cell line-derived neurotrophic factor promotes striatal neurogenesis after stroke in adult rats[J]. Stroke, 2006, 37(9): 2361-2367.
[5] Rakowicz WP, Staples CS, Milbrandt J, et al. Glial cell line-derived neurotrophic factor promotes the survival of early postnatal spinal motor neurons in the lateral and medial motor columns in slice culture [J]. J Neurosci, 2002, 22(10): 3953-3962.
[6] Duarte EP, Curcio M, Canzoniero LM, et al. Neuroprotection by GDNF in the ischemic brain[J]. Growth Factors, 2012, 30(4): 242-257.
[7] Liu YN, Wang SW, Luo SJ, et al. Intravenous PEP-1-GDNF is protective after focal cerebral ischemia in rats[J]. Neuroscience Lett, 2016, 617: 150-155.
[8] Yang CX, Zhou L, Gao XQ, et al. Neuroprotective effects of bone marrow stem cells overexpressing glial cell line-derived neurotrophic factor on rats with intracerebral hemorrhage and neurons exposed to hypoxia/reoxygenation[J]. Neurosurgery, 2011, 68(3): 691-704.
[9] Qin C, Fan WH, Liu Q, et al. Fingolimod protects against ischemic white matter damage by modulating microglia toward M2 polarization via STAT3 pathway[J]. Stroke, 2017, 48(12): 3336-3346.
[10]Wang Y, Liu G, Hong D, et al. White matter injury in ischemic stroke[J]. Prog Neurobiol, 2016, 141: 45-60.
[11]Wang X, Min S, Xie F, et al. Glial cell-derived neurotrophic factor alleviates sepsis-induced neuromuscular dysfunction by decreasing the expression of γ- and α7-nicotinic acetylcholine receptors in an experimental rat model of neuromyopathy[J]. Biochem Biophys Res Commun, 2018, 496(2): 260-266.
[12]Blesch A, Tuszynski MH. Cellular GDNF delivery promotes growth of motor and dorsal column sensory axons after partial and complete spinal cord transections and induces remyelination[J]. J Comp Neurol, 2003, 467(3): 403-417.
[13]Zhang LQ, Ma ZW, Smith GM, et al. GDNF-enhanced axonal regeneration and myelination following spinal cord injury is mediated by primary effects on neurons[J]. Glia, 2009, 57(11): 1178-1191.
[14]Gao XQ, Deng L, Wang Y, et al. GDNF enhances therapeutic efficiency of neural stem cells-based therapy in chronic experimental allergic encephalomyelitis in rat[J]. Stem Cells Int, 2016, 2016: 1431349.
[15]Barreda-Manso MA, Yanguas-Casás N, Nieto-Sampedro M, et al. Neuroprotection and blood-brain barrier restoration by Salubrinal after a cortical stab injury[J]. J Cell Physiol, 2017, 232(6): 1501-1510.
[16]Shimizu F, Sano Y, Saito K, et al. Pericyte-derived glial cell line-derived neurotrophic factor increase the expression of claudin-5 in the blood-brain barrier and the blood-nerve barrier[J]. Neurochem Res, 2012, 37(2): 401-409.
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