论文部分内容阅读
BACKGROUND: According to previous studies, the neuroprotective effect of the pedicled greater omentum may be attributed to the secretion of neurotrophic factors and stimulation of angiogenesis. The neurotrophic factors released from the pedicled greater omentum, such as brain-derived neurotrophic factor and neurotrophin 3/4/5 could exert a neuroprotective effect on the damaged host neural and glial cells, and also could induce the transdifferentiation of transplanted bone marrow mesenchymal stem cells (BMSCs) into neural cells. OBJECTIVE: Based on the functions of the omentum of neuro-protection and vascularization, we hypothesize that the transplantation of BMSCs and pedicled greater omentum into injured rat spinal cord might improve the survival rate and neural differentiation of transplanted BMSCs and consequently gain a better functional outcome. DESIGN, TIME AND SETTING: A randomized, controlled animal experiment. The experiments were carried out at the Department of Anatomy, the Secondary Military Medical University of Chinese PLA between June 2005 and June 2007.MATERIALS: Fifteen male inbred Wistar rats, weighing (200±20) g, provided by the Experimental Animal Center of the Secondary Military Medical University of Chinese PLA were used and met the animal ethical standards. Mouse anti-BrdU and mouse anti-NF200 monoclonal antibody were purchased from Boster, China. METHODS: Cell culture: We used inbred Sprague-Dawley rats to harvest bone marrow for culture of BMSCs and transplantation to avoid possible immune rejection. BMSCs were cultured via total bone marrow adherence. Experimental grouping and intervention: The rats were randomly divided into a control group, cell group and combined group, five rats per group. Rats in the control group underwent spinal cord injury (SCI) only, during which an artery clamp with pressure force of 30 g was employed to compress the spinal cord at the T10 level for 30 seconds to produce the SCI model. 5 μL PBS containing 105 BMSCs was injected into the injured site of the spinal cord in 60 seconds via a microsyringe in the cell group after SCI. In the combined group, after SCI and BMSC transplantation, an autograft pedicled greater omentum was transplanted onto the injured site of the spinal cord and fixed with a suture. SCI model and transplantation: Control group, SCI model without treatment; cell group, transplantation of BMSCs after SCI; combined group, combined transplantation of BMSCs and pedicled greater omentum after SCI.MAIN OUTCOME MEASURES: At days 1, 7, 14, 21 and 28 PO (post operation), the Basso, Beattie and Bresnahan (BBB) scale was used to observe and evaluate the recovery of locomotor function. At day 29 PO, after transcardial perfusion using 4% paraformaldehyde, a spinal cord segment of 1 cm around the injury was harvested. A cryostat section was performed longitudinally in the horizontal plane and sections were chosen by systematic random sampling for staining. Anti-BrdU staining and counting was performed to measure survival rate of transplanted BMSCs; anti-BrdU-nestin and BrdU-glial fibrillary acidic protein (GFAP) double staining and counting measured neural differentiation of BMSCs; and anti-NF 200 staining was used to evaluate axonal regeneration. RESULTS: All 15 rats were included in the outcome analysis, without any loss. Changes in BBB scores: Combined transplantation of BMSCs and the pedicled greater omentum produced significantly higher BBB scores at 7-28 days post-injury than in the control group (P < 0.05). BBB scores in the cell group were higher than in the control group at 28 days post-injury (P < 0.05). Survival rate and neural differentiation of transplanted BMSCs: Immunostaining of BrdU demonstrated that transplanted BMSCs survived in the spinal cord and migrated cranially and caudally as far as 0.5 mm from the injection site in the cell group and combined group. Some of the transplanted BMSCs expressed nestin or GFAP which revealed neural differentiation of BMSCs in the combined group and cell group. Axonal regeneration: The areas of axonal NF200 staining in the cell group and control group were lower than that of the combined group (P < 0.01).CONCLUSION: It is effective and feasible to transplant BMSCs with the pedicled greater omentum for regeneration of spinal cord after SCI compared with transplanting BMSCs alone. This method results in better locomotor outcomes and axonal regeneration.