Therapeutic Study Effect Of Co-graft Of Olfactory Ensheathing Cells With Neural Stem Cells On Rat Model With Traumatic Brain Injury

PART One Effect of olfactory ensheathing cells on proliferation and differentiation of neural stem cells in primary culture.【Objective】To explore the effect of olfactory ensheathing cells(OECs) on the proliferation and differentiation of neural stem cells(NSCs).【Methods】The cells from the embryonic rat brains were primarily cultured and identified by Immunofluorescence and immunocyochemistry method. The cells will be abserved everyday.The effect of olfactory ensheathing cells on proliferation and differentiation of neural stem cells in primary culture were abserved by comparing the experimental group and the control group.NSCs in the two-chambe shared medium with OECs were cultured in two adjacent holes in the experimental group.The proliferation of NSCs were abserved in maintenance medium at 1-7day.Simultaneously,NSCs and OECs were co-cultured in differential medium in the other corning costar . The differentiation of NSCs were abserved by Immunofluorescence and immunocyochemistry method at 4day and 7day.NSCs were cultured alone in the control group and the factors were the same as the experimental group.The datas were shown by means士standard deviation and using SPSS 13. 0 statistic software to deal with data and make 0.05 the boundary of statistical significance.【Results】Nerve growth factor receptor(P75NGFR) was observed in the primarily cultured OECs;nestin was expressed in the primarily cultured neurosphere and the cells differentiated from neurosphere expressed neurofilament 200(NF200) and glial fibrillary acidic protein(GFAP) .In the proliferation experiment,the datas of two groups were not statistical significance in first three days ,but from the 4th day the number of NSCs was increased significantly in the experimental group compared with that in the control group (P<0.05).The number of NSCs in the experimental group was on the top,then decreased and the cells were adherence and polarization.On the 4th and 7th day of differentiation,the percentage of NF200-positive cell was higher in the experimental group than that in the control group (P<0.05),indicating that the appearance of OECs increased the differentiation of NSCs into NF200-positive cells.【Conclusion】OECs can promote the proliferation of NSCs and induce the differentiation of NSCs into neurons.PART Two Effect of co-graft of Olfactory ensheathing cells with Neural stem cells on rats with traumatic brain injury.【Objective】To investigate the functional recovery of traumatic brain injury when co-transplanting NSCs and OECs in rat brain injury.【Methods】Eighty male SD rats in a SPF grade were selected.TBI model of rats in the motor cortex by electric micro-drilling was established referencing to Wang Zhanxiang'method.Sixty-four of rats were randomly divided into 4 groups(n=16 in each group),group A(NS transplantation group),group B(NSCs marked with Brdu transplation group,4×104/ul),group C(OECs transplation group,4×104/ul),group D(NSCs 40×104 and OECs 40×104 co- transplation group) .Three rats were killed in each group at 3rd,7th,14th and 30th day after transplantation and the rest was killed at 30th day.The migration,survival,differentiation of NSCs were investigated with immunofluorescene staining.On the 30th day after the operation,the number of Brdu/NF200-positive cell and Brdu-positive cell was compared in the NSCs group and co-graft group by immunofluorescene staining.Functional recovery of injury brain was assessed by Bederson scores.Using SPSS 13. 0 statistic software to deal with data and make 0.05 the boundary of statistical significance.【Results】1. In 80 SD rats,16 rats were removed and 64 rat models with TBI were successful.The scores of nervous functional def iciency in all groups were beyond 2 after operation,and all tended to decrease after the transplantatio,including the control group.In the control group,the score at the 30th day after operation was significantly decreased as compared with that at the 3rd day(P<0.05);The scores in NSCs,OECs and co-graft groups at the 14th and 30th were all significantly decreased as compared with that at the 3rd day(P<0.05);The score in the co-graft group was significantly decreased compared with that in NSCs and OECs group at the 30th day(P<0.05) .2. The cellular figure was damaged seriously and exhibited irregular cell morphology.The damage area was edema with obscured structural outlines.NSCs were located on the side of the injection.The number of cells decreased gradually away from transplanted area.After 7 days,transplanted cells migrated around the injury site and the tissue was gradully repaired.3. The result of immunofluorescene staining show the green ectogenesis BrdU positive cells were round and oval monocytes.Brdu-positive cells were seen aroud the injury site,but at 14th day the cells obviously migrated into other area away from the transplanted site.In contral group and OECs group,Brdu -positive cells were not found at the injury site.4. Few NF200-positive cells were seen around the injury site in contral and OECs group.Compare with the contral and OECs group,more NF200-positive cells and Brdu-positive cells were found on the side of the injection in NSCs and co-graft group.At 30th day,the number of Brdu-positive cell and Brdu/NF200-positive cell was increased significantly in the co-graft group (P<0.05) compared with that in the NSCs group,indicating that the appearance of OECs promoted migration,survial and differentiation of NSCs.【Conclusion】In vivo co-transplantation of NSCs and OECs can promote migration,survival and differentiation of NSCs as well as functional recovery of injury brain.