The experimental research of hypoxia-regulated NGF modified neural stem cells in acute spinal cord injury
10.3760/cma.j.cn121113-20191028-00443
- VernacularTitle:氧调控性神经生长因子基因修饰神经干细胞移植治疗急性脊髓损伤
- Author:
Mengji CHEN
1
;
Jiahui YE
;
Yibo YING
;
Min CHEN
;
Haicheng DOU
;
Wenfei NI
;
Sipin ZHU
Author Information
1. 温州医科大学附属第二医院骨科 325000
- From:
Chinese Journal of Orthopaedics
2020;40(10):669-679
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the feasibility of transplantation of neural stem cells (NSCs) modified by hypoxia-regulated nerve growth factor (NGF) gene to treat acute spinal cord injury (SCI) and observe the functional repair after SCI.Methods:Adeno-associated virus (AAV) was used as the vector to construct gene-modified NSCs. Three days after SCI attack on the animal model, the NSCs modified by hypoxia-regulated NGF were transplanted to the site of SCI as the NGF group. The GFP-modified neural stem cell group (GFP group), sham group, SCI group were set up. Hindlimb motor function was assessed by Basso-Beattie-Bresnahan (BBB) Locomotor Rating Scale, inclined plane tests and footprint analysis at 10 time points on day 1, 3, 7, 10, 14, 21, 28, 35, 42 and 60 after transplantation. The video cassette recorder (VCR) image and quantitative measurement of the height of the rat from the ground, the foot error and plantar steps were used to test the hindlimb support and flexibility of the rats. The degree of spinal cord injury in rats was roughly measured by observing the visual map of the spinal cord. The neuronal repair and morphological changes in SCI area were evaluated by Nissl staining, HE staining and immunofluorescence. CM-DiI was used to trace neural stem cells and to analyze the differentiation of NSCs by immunofluorescence.Results:Two months after transplantation of genetically modified NSCs, the BBB, inclined plane tests and footprint Analytical scores of NGF group rats were higher than those of SCI group and GFP group ( P<0.05); Through VCR image analysis, the hindlimb support and mobility of the rats in the NGF group were better than those in the SCI group and GFP group, and the difference was statistically significant ( P<0.05). Visual analysis showed that the spinal cord of the rats in each group was visually compared to the NGF group, and the spine did not show significant atrophy and color deepening, and the degree of injury was lower than that of the SCI group and GFP group; Through Nissl staining, HE staining and immunofluorescence detection, obviously positive in NeuN at the transplant site was noted at NGF group, and evidently regenerated neural structure can be seen at the morphological level. The cavity in SCI was obviously reduced, neurons and Nissl bodies were distinctly increased ( P<0.05). CM-DiI was used to track NSCs, NeuN was used to mark neurons, and GFAP was used to mark astrocytes. It was found that neural stem cells could differentiate into neurons and astrocytes. Neural stem cells in GFP group were more differentiated into astrocytes, and neural stem cells in NGF group were more differentiated into neurons. Conclusion:NSC transplantation with oxygen-regulated NGF gene mediated by adeno-associated virus can treat SCI, NSCs can differentiate into neural stem cells and astrocytes to fill the damaged cavity, NSCs secrete NGF as the carrier, playing the protective role on adjacent damaged nerve cells and reducing the death of neurons, which is expected to provide new ideas for the treatment of acute spinal cord injury, and at the same time make new attempts for the development of NGF protein drugs.
