YE Jintao,CHENG Bin,FAN Liying.Effect of GDNF gene transfection of BMSC on axon regeneration after spinal cord injury[J].Chinese Journal of Spine and Spinal Cord,2019,(6):556-564.
Effect of GDNF gene transfection of BMSC on axon regeneration after spinal cord injury
Received:February 24, 2019  Revised:May 26, 2019
English Keywords:Spinal cord injury  Bone marrow mesenchymal stem cells  Glial cell-derived neurotrophic factor
Fund:陕西省社会发展科技攻关项目(编号2016SF-187);西安市科技计划项目[编号2017115SF/YX009(13)]
Author NameAffiliation
YE Jintao Department of Orthopaedics, Xi′an Jiaotong University Second Affiliated Hospital, Xi′an, 710004, China 
CHENG Bin 西安交通大学第二附属医院骨科 710004 西安市 
FAN Liying 西安交通大学第二附属医院骨科 710004 西安市 
吴 玮  
张格林  
沈维燕  
薛建利  
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English Abstract:
  【Abstract】 Objectives: To explore the effect of bone marrow derived mesenchymal stem cells(BMSCs) transfected with glial cell line-derived neurotrophic factor(GDNF) on axonal regeneration after spinal cord injury in rats. Methods: Bilateral femurs of SD female rats were collected, and the medullary cavity was rinsed to isolate and culture BMSCs. The cell surface markers CD29, CD90, CD34 and CD45 were detected by using fluoresce in labeling technology to confirm the isolation of mesenchymal stem cells. To construct GDNF overexpressed lentiviruses to infect BMSCs, the recombinant lentiviral suspensions with infection fractions of 10, 50, 80, 100, 150 and 200 were added. Green fluorescent protein(GFP) expression was observed under fluorescent microscope to evaluate transfection cells expressing effect, GDNF expression in BMSCs after transfection was detected by Western blot. BMSCs transfected with GDNF gene were detected by MTT assay. Fifty SD rats were used to establish spinal cord blow injury model by using Allen′s blow method. After successful modeling, they were randomly divided into three groups: group A was pure BMSCs group, and BMSCs without transfection were transplanted with microsyringe; group B was the BMSCs transfection group, and BMSCs transfected with GDNF gene were transplanted with microsyringe; group C was the model control group, and DMEM medium was injected into spinal cord after modeling. BBB motor function score was performed on 7, 14 and 28 days after modeling. HE staining and microscopic observation were performed after anesthesia and perfusion. The expressions of GFAP, NSE and NF-200 in spinal cord were observed by immunohistochemical staining to evaluate the growth of axons. Results: BMSCs were isolated and cultured in a long spindle shape and grew in a fibroblast-like colony. The results of flow cytometry showed that CD29 and CD90 were highly expressed in BMSCs, while CD34 and CD45 were not expressed, indicating that the cells obtained in this study were mesenchymal stem cells. When MOI=100, BMSCs significantly expressed GFP at 12 hours after transfection, indicating successful transfection. Western blot results showed that GDFP was well expressed. MTT assay showed that the cell viability of BMSCs group was significantly higher than that of untransfected BMSCs group at 7 days after transfection. At 7 days after surgery, there was no significant difference in BBB score among the three groups. At 14 days after surgery, the BBB score of 5.80±0.19 in group B was significantly different from 3.60±0.18 in group A and 3.10±0.14 in group C. At 28 days after surgery, the BBB score of 11.90±0.28 in group B was significantly different from 8.30±0.29 in group A and 5.78±0.18 in group C (P<0.05). The optical density statistics of GFAP, NSE and immunohistochemical staining showed significant differences between group A and group B, and group B was significantly better than group A, with statistical significance(P<0.05). The statistical results of NF-200 nerve fiber length suggested 89.98±28.31μm in group B and 23.64±13.45μm in group A, with significant differences between these two groups(P<0.05). Conclusions: GDNF can successfully transfect BMSCs. The BMSCs have high cell viability after transfection. Transfection of GDNF gene can enhance the ability of BMSCs to promote axonal regeneration.
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