Investigation And Evaluation Of The Therapeutic Action Of BMSCs Combined With BMP7 In The Repair Of SCI In Adult Rat

Part 1 Experimental Study on the action of BMP7 in the differentiation of Bone marrow stromal cells Department of Orthopaedics, TongJi Hospital, Tongji Medical College, Huazhong University of Science and Technology Doctoral Candidate:Li Kuanxin Doctoral Supervisor：Prof. Li Feng AbstractObjective:1. To investigate the way of isolation, culture and induction of bone Marrow stromal cells (BMSCs) of rat in vitro.2. To explore the suitable condition, by which the BMSCs can be induced into neuron-like cells with BMP7.3. To evaluate the effect of Neuritin by observing the morphology and the molecular marker of the neuron-like induced with BMP7.Method:1. The BMSCs of rat were cultured and purified with the method of adherence of all Bone marrow cells.2. CD44,CD29,CD90,CD34andCD45 of rat BMSCs were detected by immunofluorescence technology.3. The positive rate of neuron-like cells and cell vitality were detected at 3d,1w,2w and 3w in order to build the suitable condition of induction. 4. Neuron marker (NSE), neuron dendrite marker (MAP-2), astrocyte marker (GFAP) of neuron-like cells derived form BMSCs wrer detected by immunofluorescence technology and Western blot.Result:1. The simple and efficient culture condition of BMSCs was set up with the method of adherence of all Bone marrow cells in our laboratory conditions.2. The results of immunofluorescence showed that the BMSCs cultured in our laboratory conditions expressed the BMSCs molecular marker (CD29、CD44、CD90),and did not express hematopoietic stem cell marker(CD34,CD45).3. The BMSCs were induced with BMP7 in different concentrations and different time, The positive rate of neuron-like cells and cell viability test results showed that the positive rate of neuron-like cells in group 25ng/ml BMP7 was less than the group of 50ng/ml BMP7. Above 50ng/ml BMP7, the positive rate of neuron-like cells was decreased as BMP7 concentration increasing. The positive rate of neuron-like cells reached the top after induced with 50ng/ml BMP7 for 1w. The cell viability was decreased as the time extended. The cell viability of induced group was all above 82%. After 1w, the cell viability of the group of 50ng/ml BMP7 was better than the control. So the BMSCs induced with 50ng/ml BMP7 for 1w can achieve the suitable results.4..Immunofluorescence and Western blot showed that the express of the neuron marker (NSE) and neuron dendrite marker (MAP-2) were detected,in the BMCs but not the astrocyte marker (GFAP),1W after 50mg/ml BMP7 induction.Conclusion:1. The culture and separation condition of BMSCs was set up in our laboratory, and the BMSCs of rat could be maintained in a good growth capacity and high purity.2. BMP7 can induce BMSCs into neuron-like cells expressing neuron-specific marker.PartⅡExperimental Study on the action of BMP7 in the repair of acute SCI in adult rat Objective:To establish better reasonable animal model of acute complete spinal cord injury.For investigation and evaluation of the therapeutic action BMP7 protein in the repair of spinal cord injury(SCI)in adult rats.Try to figure out the mechanism of action of BMP7 for the reparation of spinal cord injury and to search the new and effective therapeutic methods for it.Methods:The T10 spinal cord of adult rats were completely injured with the modified Allen’s method and divided into 4 groups.After injury, the injured cords were injected with Dubecco-modified Eagles Medium(DMEM,GroupⅠ) and BMP7 Protein(GroupⅡ).4w, 8w,after injury, rats were subjected to behavioral tests by using the open-field BBB scoring system and Rivlin and Tator Test. At the same time,rats were subjected to spinal induced electric potential(MEP) for evaluation therapic efficacy of spinal cord injury.Rats were sacrificed and their injured cord tissues were prepared for hematoxylin-eosin(HE) staining to detect the change of the cavity volume of histology. And for immunohistochemical staining to detect the expression levels of the neuron markers in different groupsResults:4w after transplantation,the increased levels of expression NSE,MAP-2 and GFAP were observed, and the decreased after 8w.4w after transplantation,the increased levels of expression NSE,MAP-2 and GFAP were observed, and the decreased after 8w.4w after transplantation,immunohistochemical staining showed that the transplanted cells partially were positive results and there have obviously expressed NSE.With disappearance of time, meanwhile, significant reduction in the lesion area of spinal cord tissues(P<0.05) and improvement in BBB locomotor rating scale and Rivlin and Tator Test(P<0.05)were found in the cases that received manifest improvement compared with control group.The combined transplantation treatment group have more significant change above-mentioned.Conclusions:BMP7 can obviously promote regeneration of the axone of the damage area of spinal cord tissues and improve the motor functional recovery of the animal model...