| Spinal cord injury(SCI) is very common and devastating trauma of nervous system in the clinical medical work, which always leads to severe motor and sensory dysfunction. However, there is no effective clinical treatment in the current medical conditions. Gene modified bone mesenchymal stem cells therapy of spinal cord injury is a hot spot in current research. Neurotrophic factors play a crucial role in spinal cord injury recovery. The present study attempts to explore a new way of spinal cord injury treatment. In the presnt study, bone marrow mesenchymal stem cells were transduced by lentivirus containing neurotrophin-3 gene and labeled with superparamagnetic iron oxide particles, and then transplanted into rat model with spinal cord injury via subarachnoid administration. The engrafted bone marrow mesenchymal cells overexpressed neurotrophin-3 in vivo, and more engrafted cells migrated into the lesion of injured spinal cord because homing ability of engrafted cells was improved significantly by magnetic targeting cells delivery system. So, not only the ability of bone marrow stem cells was strengthened in repairing injured spinal cord, but also the efficiency was improved. Therefore, functional recovery was further promoted and the therapeutic effect was improved. Meanwhile, the distribution of engrafted superparamagnetic iron oxide labeled stem cells in vivo could be observed by MRI.Part Ⅰ Isolating, culturing, passaging and identifying rat bone marrowmesenchymal stem cells in vitroObjective:To provide cells for the following experiments, bone marrow mesenchymal stem cells of rats were isolated and purified using the method of whole bone marrow adherent, and morphology, surface marker and proliferation capability of isolated cells were identified after cultured and passaged.Methods:1. Bone marrow mesenchymal stem cells of rats were isolated and purified using the method of whole bone marrow adherent, and the isolated cells were cultured and passaged, meanwhile cell morphology was evaluated with microscopy. 2. To analyzed characteristic of the isolated cell growth, the growth curves were tested by MTT assay. 3. To identify whether the isolated and cultured cells have characteristics of bone marrow mesenchymal stem cell, the surface markers(CD29, CD34, CD44, CD45) of bone marrow mesenchymal stem cells were detected by flow cytometry.Results:1. rat bone marrow mesenchymal stem cells that were isolated and purified using the method of whole bone marrow adherent were fusiform and could be cultured and passagedin vitro. 2. The growth curves of cells at passage 3, 4 and 5 were S-shape and similar which were tested by MTT assay, which indicated the cells have enormous proliferative capacity. 3. By flow cytometry detection of cells surface markers, expressions of CD29 and CD44 were positive, while expressions of CD34 and CD45 were negative, which corresponded to the general characteristics of bone marrow mesenchymal stem cell.Conclusion:The method of whole bone marrow adherent is simple, economical and practical, highly efficient, which is able to isolated and purified rat bone marrow mesenchymal stem cells. The isolated cells have enormous proliferative capacity, can be repeatedly passaged and meet of research requirements.Part Ⅱ neurotrophin-3 gene transfecting and superparamagnetic iron oxide particles labeling of rat bone marrow mesenchymal stem cellsObjective:For preparation of cell transplantation combined magnetic targeting, bone marrow mesenchymal stem cells were transfected by lentivirus containing neurotrophin-3 gene to stably overexpress neurotrophin-3 protein and labeled with superparamagnetic iron oxide particles.Methods:1. att B1-NTF3-att B2 was amplified by overlapping PCR, p Down-NTF3 was constructed using Gateway technology, plasmid(p LV.EX3 d.P / puro-EF1α> NTF3> IRES / Ds Red Express2) containing neurotrophin-3(NT-3) and red fluorescent protein(Ds Red) gene was constructed with Gateway technology, finally lentiviral packaging plasmid was performed. 2. NTF3-Ds Red gene was transduced into bone marrow mesenchymal stem cells with lentivirus vector. 3. NT-3 m RNA expression of NTF3-Ds Red gene transfected bone marrow mesenchymal stem cells was detected by Real-time PCR. 4. NT-3 protein expression of NTF3-Ds Red gene transfected bone marrow mesenchymal stem cells was detected by western blotting. 5. After superparamagnetic iron oxide(SPIO) particles with concentration of 25 μg/ml labeled NTF3-Ds Red gene transfected rat bone marrow mesenchymal stem cells, the ratio of labeled cells was tested by Prussian blue staining.Results:1. Th constructed plasmid(pLV.EX3 d.P / puro-EF1α> NTF3> IRES / DsRed Express2) containing NT3-Ds Red gene was correct by sequencing genes, which transfected 293 FT cells successfully. The titers of the packaged lentivirus was about 1.4×108TU/ml.2. Red fluorescence emitted by NT3-Ds Red gene transfected bone marrow mesenchymal stem cells could be observed under fluorescence microscope. 3. Real-time PCR analysis showed NT-3 m RNA expression level of NT3-Ds Red gene transfected bone marrow mesenchymal stem cells was 13 times that of NT3 gene untransfected bone marrow mesenchymal stem cells. 4. Western blotting analysis showed NT3-Ds Red gene transfected bone marrow mesenchymal stem cell were able to overexpress NT-3 protein, while NT3 gene untransfected cells did not express NT-3 protein. 5. After SPIO with concentration of 25 μg/ml labeled NT3-Ds Red gene transfected bone marrow mesenchymal stem cells successfully, Prussian blue staining showed cytoplasm was blue, nucleus was pale red and the ratio of labeled was 100%.Conclusion:The constructed lentivirus vector containing NT3-Ds Red gene was able to transducedNT3-Ds Red gene into the DNA of rat BMSC, so that BMSC could efficiently and stably overexpress NT-3 factor. Meanwhile, SPIO with concentration of 25 μg/ml could effectively label NT3-Ds Red gene transfected bone marrow mesenchymal stem cells in vitro. Laid foundation for the further in vivo experiments.Part Ⅲ Effects of magnetical targeting NT-3 gene transfected bone marrow mesenchymal stem cells in a rat model of spinal cord injury and MR imaging in vivoObjective:To evaluate the injured spinal cord tissue repair, hindlimb motor function recovery and therapeutic effect, SPIO-labeled and NT3 gene transfected bone marrow mesenchymal stem cells were transplanted by subarachnoid administration and delivered into the lesion of injured spinal cord by a magnetic targeting system. Meanwhile, the engrafted cells were imaged by MR for observing cells distribution in the rat body.Methods:1. Aftere laminectomy was performed on the T7-8 vertebrae and the dura mater was exposed, SD rat model of spinal cord injury was induced by a weight-drop device. 2. On days 7 after spinal cord injury, 36 rats were randomly assigned into three groups: ①BMSCs group, n=12, Ds Red gene transfected BMSCs were injected into cerebrospinal fluid; ②NT3 group, n=12, NT3-Ds Red gene transfected BMSCs were injected into cerebrospinal fluid; ③M-NT3 group, n=12, after NT3-Ds Red gene transfected BMSCs were injected into cerebrospinal fluid, a slab of neodymium magnet was attached to the spine of the rat at the T7 level and removed after 24 h. 3. After the magnet was removed, MR imaging was performed in order to observe the engrafted cells distribution in the rat body. 4. After transplantation, hind-limb motor function of all the rats were evaluated according to BBB scale(Basso,Beattie,Bresnahan locomotor rating scale)and the averaged BBB scores were recorded on days 1, 3, and 7, and then every week up to the fifth week. 5. On days 35 after transplantation, all the rats were euthanized to harvest the injured spinal cord tissues. The expressions of NT3 protein in the injured spinal cords were assessed by western blotting, the cystic cavity area in the lesion was assessed by HE staining, SPIO-labeled cells in the injured spinal cords were detected by Prussian blue staining, the expressions of neurofilament protein(NF200) and glial fibrillary acidic protein(GFAP) around the lesion were evaluated by immunofluorescence labeling. 6. All data are expressed as the mean(x—) ± standard deviation(SD). The BBB score data were analysed with repeated measures analysis of variance(ANOVA). All other data were analyzed using one-way ANOVA to identify significant differences among the three groups. Statistical significance was inferred when p < 0.05. All statistical analyses were performed using SAS 6.12 and SPSS 17.0.Results:1. MR imaging of rats with acute spinal cord injury in each group which underwent BMSCs transplantation showed that the signal intensity(SI) of the injured spinal cords in the M-NT3 group decreased significantly compared with those of NT3 groups, whereas no apparent SI change was observed in the BMSCs group. 2. The BBB scores of rats with acute spinal cord injury in each group showed that the BBB scores of the M-NT3 group did not improve significantly compared with those of the other two groups on days 1 and 3 after cell transplantation, but the BBB scores of the M-NT3 group were the highest among the three groups on days 7, 14, 21, 28, and 35 after cell transplantation, the BBB scores of the NT3 group were also significantly higher than those of the BMSCs group on days 7, 14, 21, 28, and 35 after cell transplantation. Moreover, significant differences(n=12, p<0.05) were observed among the three groups on days 7, 14, 21, 28, and 35 after cell transplantation, whereas no significant differences(n=12, p>0.05) were observed among the three groups on days 1 and 3 after cell transplantation. 3. On day 35 after cells transplantation, western blotting analysis showed that NT3 protein expression level in the M-NT3 group was significantly higher than that in the NT3 group and BMSCs group, whereas the NT3 protein level in the BMSCs group was significantly lower than that in the NT3 group. Significant differences(n=12, p<0.05) were observed among the three groups using one-way ANOVA analysis. 4. On day 35 after cells transplantation, Prussian blue staining of the injured spinal cord tissues indicated significantly more blue-stained cells were observed in the M-NT3 group than in the NT3 group, whereas no blue-stained cells could be observed in the BMSCs group 5. On day 35 after cells transplantation, HE staining of the injured spinal cord tissues showed the mean values of the cystic cavity area in the BMSCs, NT3, and M-NT3 groups were 0.64±0.14 mm2, 0.51±0.11 mm2 and 0.39±0.10 mm2(x—±SD), respectively. Statistically significant differences were observed among the three groups(n=12, p<0.05) using one-way ANOVA analysis. 6. On day 35 after cells transplantation, immunofluorescence labeling of the injured spinal cord tissues showed the NF200 expression level of the M-NT3 group was hightest and the BMSCs group was lowest among the three groups, whereas the GFAP expression level of the M-NT3 group was lowest and the BMSCs group was highest among the three groups. Statistically significant differences were observed among the three groups(n=12, p<0.05) using one-way ANOVA analysis.Conclusion:Magnetic targeting SPIO-labeled and NT3 gene transfected bone marrow mesenchymal stem cells for therapy of spinal cord injury not only could greatly improve the efficiency of cells homing, but also could significantly reduce cystic cavity area of injured spinal cord, promote axonal regeneration, inhibit glial scar formation and improve functional recovery. Meanwhile, engrafted bone marrow mesenchymal stem cells could also be imaged by MR in vivo, which could help to observe the distribution of engrafted cells in the body. This method of spinal cord injury therapy is highly efficient, minimally invasive and promising for the clinical application. |
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