[Preliminary study on effects of human brain-derived neurotrophic factor gene-modified bone marrow mesenchymal stem cells by intravenous transplantation on structure and function of rat injured spinal cord].
OBJECTIVE
To transplant intravenously human brain-derived neurotrophic factor (hBDNF) gene-modified bone marrow mesenchymal stem cells (BMSCs) marked with enhanced green fluorescent protein (EGFP) to injured spinal cord of adult rats, then to observe the viability of the cells and the expressions of the gene in spinal cord, as well as the influence of neurological morphological repairing and functional reconstruction.


METHODS
Ninety-six male SD rats weighing (250 +/- 20) g were randomly divided into 4 groups: hBDNF-EGFP-BMSCs transplantation group (group A, n = 24), Ad5-EGFP-BMSCs transplantation group (group B, n = 24), control group (group C, n = 24), and sham operation group (group D, n = 24). In groups A, B, and C, the spinal cord injury models were prepared according to the modified Allen method at the level of T10 segment, and after 3 days, 1 mL hBDNF-EGFP-BMSCs suspension, 1 mL Ad5-EGFP-BMSCs suspension and 1 mL 0.1 mol/L phosphate buffered saline (PBS) were injected into tail vein, respectively; in group D, the spinal cord was exposed without injury and injection. At 24 hours after injury and 1, 3, 5 weeks after intravenous transplantation, the structure and neurological function of rats were evaluated by the Basso-Beattie-Bresnahan (BBB) score, cortical somatosensory evoked potential (CSEP) and transmission electron microscope. The viability and distribution of BMSCs in the spinal cord were observed by fluorescent inverted phase contrast microscope and the level of hBDNF protein expression in the spinal cord was observed and analyzed with Western blot. Meanwhile, the expressions of neurofilament 200 (NF-200) and synaptophysin I was analyzed with immunohi stochemistry.


RESULTS
After intravenous transplantation, the neurological function was significantly improved in group A. The BBB scores and CSEP in group A were significantly higher than those in groups B and C (P < 0.05) at 3 and 5 weeks. The green fluorescence expressions were observed at the site of injured spinal cord in groups A and B at 1, 3, and 5 weeks. The hBDNF protein expression was detected after 1, 3, and 5 weeks of intravenous transplantation in group A, while it could not be detected in groups B, C, and D by Western blot. The expressions of NF-200 and synaptophysin I were stronger and stronger with transplanting time in groups A, B, and C. The expressions of NF-200 and synaptophysin I were strongest at 5 weeks, and the expressions in group A were stronger than those in groups B and C (P < 0.05). And the expressions of NF-200 in groups A, B, and C were significantly stronger than those in group D (P < 0.05), whereas the expressions of synaptophysin I in groups A, B, and C were significantly weaker than those in group D (P < 0.05). Ultramicrostructure of spinal cords in group A was almost normal.


CONCLUSIONS
Transplanted hBDNF-EGFP-BMSCs can survive and assemble at the injured area of spinal cord, and express hBDNF. Intravenous implantation of hBDNF-EGFP-BMSCs could promote the restoration of injured spinal cord and improve neurological functions.
Lin
Jianhua
J
Department of Orthopedics, First Affiliated Hospital of Fujian Medical University, Fuzhou Fujian 350005, PR China. jianhual@126.com
Wang
Changsheng
C
Wu
Zhaoyang
Z
D020816:Amino Acid Motifs; D000818:Animals; D001854:Bone Marrow Cells / Q000378:metabolism; D019208:Brain-Derived Neurotrophic Factor / Q000235:genetics*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D008297:Male; D045164:Mesenchymal Stem Cell Transplantation; D051381:Rats; D017207:Rats, Sprague-Dawley; D013119:Spinal Cord Injuries / Q000473:pathology* / Q000503:physiopathology* / Q000601:surgery
D019208:Brain-Derived Neurotrophic Factor; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins