组织工程的提出、建立和发展,为最终实现脊髓损伤的修复和真正意义上的结构、形态与功能重建开辟了新的途径。支架的生物活性、三维结构和表面微观结构,材料的降解性等众多因素都对细胞增殖,分化和组织形成有明显影响。组织工程的发展也将改变传统的医学模式,使得再生医学得以进一步发展,并最终用于疾病的治疗。
To summarize Notch, basic hel ix-loop-hel ix (bHLH) and Wnt gene signal transduction pathways in the process of differentiation and development of neural stem cells. Methods The l iterature on the gene signal transduction pathway in the process of differentiation and development of neural stem cells was searched and then summarized and analyzed. Results The formation of Nervous System resulted from common actions of multi-signal transduction pathways. There may exist a fixed threshold in the compl icated selective system among Notch, bHLH and Wnt gene signal transduction pathways. Conclusion At present, the specific gene signal transduction pathway of multi pl ication and differentiation of neural stem cells is still unclear.
Objective To observe the effect of transplantation of embryonic stem cell(ES) on neurological functional recovery of injured spinal cord in adult mouse. Methods The ES cells were cultured and induced in vitro. Fifty C57/BL6J mice were made animal model of semicut mice of T9，10. The ES cellderived neural precursors cells were transplanted into the vertebral canalaround injured spinal cord semi-cut mice. Twenty-eight C57/BL6J mice were randomly divided into three groups: sham operation group(group A,n=9), operation／cell group (group B，n=10), and operation／DMEM group(group C,n=9). RT-PCR analysis, X-gal staining and immunofluorescence were used to observe the cells survival and differentiation in the spinal crod. BBB test was performed to study functional improvement. Results ES cells induced and cultured in vitro displayed clonal growth with circle or ovoid shape and had one or more nucleoli. RT-PCR result showed that the induced ES cells expressed mRNA of Nestin and microtubuleassociated protein, but did not express glial fibrillory acidic protein(GFAP). There was statistically significant difference in BBB scoring between group A and groups B, C after operation (P＜0.01). There was statistically significant difference in BBB scoring at 1, 2 and 4 weeks of operation(P＜0.01), but no statistically significant difference at 6 and 8 weeks of operation between groups B and C(P＞0.05). The X-gal staining results werepositive in group B and negative in groups A and C. The immunoflurescence resultshowed neurofilament green fluor and no expression of GFAP in injured spinal cord region. Conclusion After transplantation, ES cellderived cells can survive, transfer into the injury position, and differentiate into neurons, but spinal cord function has no obvious improvement.
ObjectiveTo investigate the expression pattern and significance of Sonic Hedgehog (Shh) signaling pathway by observing whether the Shh signaling pathway components express in the adult rat after spinal cord injury (SCI). MethodsSixty-four healthy male Sprague-Dawley rats were randomly divided into normal group (group A, 8 rats), sham group (group B, 8 rats), and SCI group (group C, 48 rats). In group A, the rats served as controls without any treatment; a decompressive laminectomy was performed on T7-9 levels without SCI in group B; and modified Allen's method was used to make SCI model in group C. Basso Beattie Bresnahan (BBB) scale was used to assess the hind limb motor function at 12 hours, 1 day, 3 days, 7 days, 14 days, and 21 days after SCI; the immunofluorescence staining, real-time PCR, and Western blot were performed to detect the mRNA and protein expression levels of Shh and Glioma-associated oncogene homolog-1 (Gli-1) in SCI zone. ResultsThe BBB score slowly increased with time in group C, but the scores at each time point in group C were significantly lower than those in group A and group B (P<0.05). The results of immunofluorescence staining showed that Shh and Gli-1 rapidly increased after SCI in astrocytes. Real-time PCR and Western blot showed that the relative expression levels of Shh and Gli-1 mRNA and protein were gradually increased in group C and reached a maximum at 7 days. In addition, the relative expression levels of Shh and Gli-1 mRNA and protein in group C were significantly higher than those in group A and group B (P<0.05). On the other hand, compared with group A, the expression of Gli-1 protein was reduced in the cytoplasm but increased in nucleus in group C. ConclusionAstrocytes synthesize and secrete Shh and Gli-1 signaling molecules after SCI, both Shh and Gli-1 significantly up-regulate and exhibit dynamic changes, which suggests Shh signaling pathway may be involved in nerve cell regeneration after SCI.
Objective To study the growth characteristics of umbil ical cord MSCs (UCMSCs) in vitro and its effect on the nerve regeneration after spinal cord injury (SCI). Methods UCMSCs isolated from pregnant rats umbil ical cord were cultured and purified in vitro. Sixty female Wistar rats weighing (300 ± 10) g were randomized into three groups (n=20per group). UCMSCs group (group A) in which UCMSCs suspension injection was conducted; DMEM control group (groupB) in which 10% DMEM injection was conducted; sham group (group C) in which the animal received laminectomy only.Establ ish acute SCI model (T10) by Impactor model-II device in group A and group B. The recovery of the lower extremity was observed using BBB locomotor scoring system, neurofilament 200 (NF-200) immunofluorescence staining was performed to detect the neural regeneration, and then the corticospinal tract (CST) was observed using the biotinylated dextran amine (BDA) tracing. Results Cultured UCMSCs were spindle-shaped fibrocyte-l ike adherent growth, swirl ing or parallelly. The USMSCs expressed CD29, but not CD31, CD45, and HLA-DR. The BBB score was higher in group A than group B 4, 5, and 6 weeks after operation, and there was a significant difference between two groups (P lt; 0.05). The BBB scores at different time points were significantly lower in groups A and B than that in group C (P lt; 0.05). UCMSCs was proved to survive and assemble around the injured place by frozen section of the cords 6 weeks after injury. NF-200 positive response area in groups A, B, and C was (11 943 ± 856), (7 986 ± 627), and (13 117 ± 945) pixels, respectively, suggesting there was a significant difference between groups A, C and group B (P lt; 0.05), and no significant difference was evident between group A and group C (P gt; 0.05). BDA anterograde tracing 10 weeks after operation demonstrated that more regenerated nerve fibers went through injured area in group A, but just quite few nerve fibers in group B went through the injuried cavity. The ratios of regenerative axons amount to T5 axons in group A and group B were smaller than that of group C (P lt; 0.05). Conclusion UCMSCs can prol iferate rapidly in vitro, survive and differentiate to neurons after being grafted into injured spinal cord. The transplantation of UCMSCs is effective in promoting functional recovery and axonal regeneration after SCI.
Objective To review the status and appl ication prospect in repair of spinal cord injury by stem cells. Methods The related articles in recent years were extensively reviewed, the biological characteristics of stem cells, the experimental and cl inical studies on repair of spinal cord injury by stem cells, the mechanism of the therapy and the problem were discussed and analyzed. Results The foundational and cl inical study indicated that the great advance was made in repair of spinal cord injury, the stem cells could immigrate in the spinal cord, and differentiate into neuron and secrete neurotrophic factors. So it could promote the repair effects. Conclusion Repair of spinal cord injury by stem cells is an effective therapystrategy, but many problems remain to be resolved.
Objective To investigate the synergistic effect of a combination of grafted olfactory ensheathing cells (OECs) from the olfactory bulbs and intrathecal injection of vascular endothel ial growth factor (VEGF) on repairing spinal cord injury, and to explore the neuroprotection on both neurons and nerve fibers. Methods OECs from neonatal rats were cultured, purified, and collected with 0.25% trypsin after 9 days. A total of 75 adult female Wistar rats (weighing 200-250 g) were randomly divided into 5 groups: group A was sham-surgery group receiving laminectomy; the spinal cord injury model was establ ished with weight-dropped apparatus in the rats of groups B, C, D, and E. Then group B was injected with 10 μL DMEM-F12 medium without serum at injury site on the 1 day and was intrathecally administrated with 10 μL sal ine solutiontwice a day during the following 1 week; group C was injected with 10 μL DMEM-F12 medium and 25 ng recombined ratVEGF165 (rrVEGF165); group D was injected with 10 μL DMEM-F12 medium containing 1 × 105 OECs and 10 μL sal ine solution; group E was injected with 10 μL DMEM-F12 medium containing 1 × 105 OECs and 25 ng rrVEGF165. The functional recovery of hindl imb was evaluated by the Basso-Beattie-Bresnahan (BBB) score at 1 day and each week from 1 to 8 weeks. The histological changes and the changes of ultrastructure were observed at 8 weeks after operation by HE and electron microscope, and the immunohistochemistry staining was used for p75 nerve growth factor receptor (p75NGFR), Caspase-3, and von Willebrand factor (vWF). Results The function of hindl imb recovered rapidly in group E; the BBB score reached the peak at 8 weeks, and it was significantly higher than those in other groups (P lt; 0.05). The histology and ultrastructure observation showed that nerve fibers and neurons were damaged seriously in group B, oderately in groups C and D, and sl ightly in group E. Numerous spared tissue between nerve stumps, fibers with regular myel ination, and neurons with l ittle vacuolar mitochondria were observed in group E. The immunohistochemistry staining revealed that Caspase-3 positive cells in groups B, C, D, and E were significantly more than that in group A (P lt; 0.05); more Caspase-3 positive cells were found in groups B and D than in groups C and E (P lt; 0.05), while no significant difference was found between groups C and E (P gt; 0.05). And more vessels per high field were examined in groups C and E than in groups A, B, and D (P lt; 0.05), while no significant difference was found between groups C and E (P gt; 0.05). The p75NGFR positive results showed the survival of OECs in groups D and E at 8 weeks after OECstransplantation. Conclusion Grafted OECs combined with intrathecal injection of VEGF has significant promotive effects on restoration of spinal cord injury in rats, can improve part function of nerve fibers, and shows neuroprotection on damaged cells and fibers, which have a synergistic effect.
OBJECTIVE: To observe the effect of nerve growth factor (NGF) and nimodipine (NP) on fetal spinal cord graft in repair of injury of spinal cord. METHODS: A total of 144 adult Wistar rats were included in this study. All were made as the hemi-section cavity injury model at the lumbar enlargement and divided into three groups: fetal spinal cord graft (group Tr), fetal spinal cord graft with NGF (group TN), and fetal spinal cord graft with NGF and NP (group TNN). The intracellular concentration of free ionic calcium was measured at the 4th, 8th, and 24th hour, and superoxidase (SOD) and malondialdehyde (MDA) at 3rd, 6th, 12th, 24th and 72nd hour after operation. RESULTS: After spinal cord was injured, the concentration of MDA and intracellular concentration of free ionic calcium increased and reached to the peak at the 6th and 8th hour respectively, but SOD decreased and at 24th hour to its vale. The MDA was significantly lower in group TN than in group Tr, while the SOD was higher (P lt; 0.05). There was no significant difference on intracellular free ionic calcium concentration between group Tr and TN. The concentration of SOD of group TNN was the highest and the intracellular concentration of free ionic calcium was the lowest in the three groups (P lt; 0.05). The weekly mortality was 33%, 31%, 17% respectively in group Tr, TN and TNN. The mortality of group TNN was significantly lower than the other two groups (P lt; 0.01). CONCLUSION: Although the fetal spinal cord graft is an effective method to repair laboratory spinal cord injury, NGF and ND can interrupt secondary injury and increase survival rate of the host.
Objective To investigate the influence of different transplantating times on the survival and immigration of the bone marrow mesenchymal stem cells (BMSCs) in injured spinal cord by subarachnoid administration, and to evaluate the most optimal subarachnoid administration times for BMSCs. Methods Eight adult male rats (weighing 120 g) were used to isolate BMSCs that were cultured, purified and labeled with Hoechst 33342 in vitro. Another 75 adult Wistar rats (weighing 220 g) were made the spinal cord injury (SCI) models at T9,10 level according to the improved Allen’s method and were randomly divided into 5 groups (groups A, B, C, D, and E, n=15). The labeled BMSCs at 1 × 107/mL 0.1 mL were injected into subarachnoid space of the rats via a catheters under the subarachnoid space in groups A (one time at 1 week), B ( two times at 1 and 3 weeks), C (3 times at 1, 3, and 5 weeks) and D (5 times at 1, 3, 5, 7, and 9 weeks) and 0.2 mL phosphate-buffered sal ine (PBS) was injected in group E (5 times at 1, 3, 5, 7, and 9 weeks) as blank control. The neurological functions were evaluated using the Basso-Beattie-Bresnahan (BBB) scale 1, 3, 5, 7, 9, and 12 weeks after transplantation. The migration, survival, differentiation, and histomorphological changes of BMSCs were observed by HE, immunohistochemistry, and fluorescence microscopy. Results At 3 weeks after injury, there were significant differences in the BBB scores between group E and groups A, B, C, D (P lt; 0.01), and between groups A, B and groups C, D (P lt; 0.01). At 7, 9, and 12 weeks, the BBB scores were significantly higher in groups C and D than in groups A and B (P lt; 0.01), and in group B than in group A (P lt; 0.01). There were no significant differences in the BBB scores between groups C and D (P gt; 0.05). The fluorescence microscopy showed that the transplanted BMSCs survived and grew in the injured region at 3 weeks after injury and as time went on, the transplanted cells gradually decreased in group A; in groups B, C, and D, BMSCs count reached the peak values at 5 and 7 weeks and then gradually decreased. At 12 weeks, the survival BMSCs were significantly more in groups C and D than in groups A and B (P lt; 0.01). HE staining showed that the formation of cavity was observed in each group at 3 weeks after injury and the area of cavity gradually decreased in groups A, B, C, and D. At 12 weeks, the area of cavity was the miximal in groups C and D, moderate in groups A and B, and the maximal in group E. The immunohistochemistry staining indicated that the expression of NF-200 was more intense in groups C and D than in groups A and B. The expression of NF-200-positive fibers was more intense in group C. Conclusion Multiple administration of BMSCs promotes the restoration of injured spinal cord and improves neurological functions, and three times for BMSCs transplantation is best
Objective To investigate the effect of olfactory ensheathing cell culture medium (OECCM) on the growth of spinal cord neurons and its protective effect on the injured neurons by H2O2, and to disscuss the probable protective mechanisms of olfactory ensheathing cells (OECs). Methods The primary olfactory ensheathing cells (OECs) were isolated from olfactory bulb of adult SD rat, and OECCM were prepared. The morphology of OECs was observed by inverted phase contrast microscope, identified by rabbit-antiratlow-affinity nerve growth factor p75 (NGFRp75), and its purity were calculated.Primary spinal cord neurons were cultured from 15 to 17 days pregnant SD rats, and injury model of neurons were prepared by H2O2. OECCM and control culture medium were added into the normal spinal neurons (groups A, B). OECCM and control culture medium were added into the injured spinal neurons by H2O2 (groups C, D). In groups A and C, 200 μL of control culture medium was used; in groups B and D, 100 μL of control culture medium and 100 μL of OECCM were used. Then the growth index such as average diameter of neuron body, the number and length of neuron axons were measured. The viabil ities of normal and injured neurons were assessed by MTT. Results OECs showed bipolar or tripolar after 6-9 days of culture. Primary spinal cord neurons were round and bigger, and neuron axons grew significantly and showed bipolar after 5-7 days of culture. The immunocytochemisty of OECs by NGFRp75 showed that membrane were stained. The degree of purity was more than 90%. Primary spinal cord neurons grew well after 6-9 days of culture, and compared with group A, neurons of group B grew b, whose cell density and diameter were bigger. The average diameter of neuron body, the number and length of neuron axons were (33.38 ± 6.80) D/μm, (1.67 ± 0.80), and (91.19 ± 62.64) L/μm in group A, and (37.39 ± 7.28) D/μm, (1.76 ± 0.82), and (121.33 ± 81.13) L/μm in group B; showing statistically significant differences (P lt; 0.05). The absorbency (A) value of neurons was 0.402 0 ± 0.586 9 in group A and 0.466 0 ± 0.479 0 in group B; showing statistically significant difference (P lt; 0.01). After 2 hours of injury by H2O2, the cell density of spinal cord neurons decreased, and neuron axons shortened. The A value of injured neurons was 0.149 0 ± 0.030 0 in group C and 0.184 0 ± 0.052 0 in group D, showing statistically significant difference (P lt; 0.01). Conclusion The results above suggest that OECCM could improve the growth of spinal cord neurons and protectthe injured neurons. The neurotrophic factors that OECs secrete play an important role in the treatment of spinal cord injury.