An Experimental Study On Repair Of Peripheral Nerve Injury By Transplantation Of BMSCs

It is an important task to promote peripheral nerve regeneration after injury in neuroscience. Recent technical progress of molecular biology,cell biology and microsurgery results in better outcome,but not all patients have satisfactory results.It is a nodus how to improve the recovery after nerve injury in peripheral nerve surgery.Bone marrow mesenchymal stem cells(BMSCs) is one kind of adult stem cells that have self-renewal and multilineage differentiation potential.It has recently been reported that BMSCs can differentiate into neural cells,opening new frontiers in therapy for nerve injury.In this study, we repaired of rat peripheral nerve injury by transplantation of BMSCs,and examined the mechanism of BMSCs transplantation for promoting regeneration of peripheral nerve. We wanted to offer the experimental data for clarifying the mechanism and clinical application.Our experimental study were divied into three parts: Part I:MSCs(mesenchymal stem cells) derived from rat bone marrow were isolated and culture expanded efficiently in vitro and its biological features are detected. We selected suitable culture medium and FBS concentration ,separated and purified the BMSCs with adherent method. The morphology of BMSCs was observed with invert-microscope constantly. BMSCs' curve of growth was depicted and cell cycle was analyzed by flowing cytometry. BMSCs were induced to differentiate into osteoblasts and adipocytes in induction medium containing desamethasone,β-Glycerophosphate and horse serum. Then differentiated cells were identificated by AKP and oil O stain. Results showed BMSCs could be isolated and proliferated by adherent method in vitro. The appearance of BMSCs were fibroblast-like cells and its growth curves was like shape of S. Cell cycle analysis showed that most of BMSCs were in G1/G0 phase. BMSCs can be differentiated into osteoblasts and adipocytes in vitro.Part II:Investigate the neural differentiation potential and expression NGF (nerve growth factor) and BDNF (brain-derived neurotrophic factor)in BMSCs. BMSCs were induced with BME(β-mercaptoethanol) in L-DMEM condition medium . The changed ultrastructure of BMSCs was observed by transmission electron microscope. Then expression of NGFmRNA and BDNFmRNA was detected by RT-PCR. Results showed a certain number of cells adopted neuron-like morphological changes and expressed NSE, but no GFAP by immunohistochemistry after　 induction. Some BMSCs had typical neurolike ultrastructure after induction. BMSCs expressed NGFmRNA and BDNF mRNA before induced.Part III:Study on repair of sciatic nerve by transplantation of BMSCs and effect of expression of NGF and BDNF. Sciatic nerve crush injury model was set up in female Wistar rats. The BMSCs labeled by BrdU were transplanted into the lesion of nerve injury by injection immediately. We detected BMSCs labeled by BrdU after transplantation and set up control group.To evaluate the repair results of peripheral nerve injury by:⑴measuring walking behavior by footprint analysis every week after operation;⑵determining conduction velocity of the regenerated sciatic nerve of two groups;⑶observing regenerated sciatic nerve fibers by Luxol Fast Blue stain and TEM at 6 weeks after operation ;⑷measuring the wet weight of the corresponding gastrocnemius and the area of the muscle fibers.The expressions of NGF and BDNF in the distal nerve segments were detected by immunohistochemistry and imaging analysis at 1,2,4 and 6 week after operation. The results were below:The BrdU-positive cells could be found until 4 weeks after operation;SFI of experimental group was better than the control group 3 week later;count and diameter of regenerated sciatic nerve fibers of experimental rats were superior to the control ,too;the weight of corresponding gastrocnemius and the area of the muscle fibers got the same result; NGF and BDNF in experimental　 group expressed positive compared with the control at 1,2week,but negative at 4,6week.Conclusion:⑴Rat BMSCs have been successfully isolated and culture-expanded in vitro with adherent method;⑵BMSCs can be differentiated into NSE postive cells in vitro.⑶BMSCs expressed NGFmRNA and BDNF mRNA before induced。⑷BMSCs can be transplantated into injury tissue and promote the recovery of nerve function in the micro-enviroment.