Experimental Study On The Effection Of Local Low Dose X-ray Radiation On Homing Of Bone Marrow Mesenchymal Stem Cells To Injured Peripheral Nerve

Part 1 The separation, culture, identification and dyeing of Bone marrowmesenchymal stem cellsObjective:To master the methods for isolation, culture, amplification and dyeing of bone marrow mesenchymal stem cells(BMSCs) of rat in vitro and explore their biological characteristics prelliminarily.Methods:Disinfection after the rat were put to death, removed the femurs and tibias of SD rat under asepsis procedure, exposed the bone marrow cavity, the BMSCs were collected by flushing the shaft with L-DMEM, centrifugation, supernatant, resuspend count in L-DMEM medium and cultured in 37 °C, Saturation humidity, 5% CO2 environment, following by applicating of whole bone marrow and adherent separation. when 85%-90%fusion, the cells were cultured with trypsin enzyme-digesting technique and passaged in vitro. The morphology of the cells was observed by phase contrast microscope. The surface markers of cells were analyzed by flow cytomety. The growth curve of cells was drawn by cell counting. Labelled the cells with CFDA SE dye.Results:A few primary cells were observed adhere to the flask within 2h after cultured,mostly round and had high refraction.One day later,the number of cell adhere to the flask increased,most of them were round or oval,a few fusiform.Three days later,the cells adhere to the flask were in irregular shape such as fusiform,polygon,some of them forming tufted cell clusters.Generally,the first two to four days the proliferation of cells was slowly,cell morphology changed to fusiform gradually,there were also a few round and polygon cell.From day 5 to day 8,the cells proliferated fastly,the number of cell clusters increased.The cells would reach 85%-90%fusion for 10 to 14 days,they arranged局部低剂量X线辐照对BMSCs向周围神经损伤部位归巢影响的实验研究英文摘要as a whirlpool regularly,it was the time to passage.The growth curve of P2,P4,P6 of the cell were similar.The surface marker of P4 generation BMSCs were analyzed by flow cytomety,the result run thus:CD34(0.8%),CD45(1.3%)negative;CD29(93.7%),CD90(99.8%)positive.Conclusion:The BMSCs from rat bone marrow can be isolation, purification and cultivation in vitro simply and quickly by applicating of whole bone marrow and adherent separation. And also the BMSCs could proliferate rapidly and keep their biological characters stable. Part 2 Experimental study on the effection of local low dose X-ray radiation on homing of bone marrow mesenchymal stem cells to injured peripheral nerveObjective:To observe the effection of local low dose X-ray radiation on homing of bone marrow mesenchymal stem cells to injured peripheral nerve combined small gap anastomosis neurorrhaphy with local low dose X-ray radiation and transplantation BMSCs via caudal vein of SD rats, and investigate the possible mechanism. Methods:Sixty two-month old Sprague-Dawley rats weighing 250g~300g were selected and randomly divided into two groups after the rat models of peripheral nerve injury established by small gap anastomosis neurorrhaphy. The experimental group was irradiated by dose of 1 Gy X-ray, while the control group was placed in the same environment, but not radiation. One hour after the radiation, all rats transplanted BMSCs via caudal veins. Each group randomly selected six rats to produce frozen sections and then observe the difference of BMSCs homing under fluorescence microscope at the time point(24h、48h、96h) after transplantation. And also evaluate the recovery of neurological function between the two groups through general state, general form of nerve under microscope, sciatic nerve functional index, motor nerve conductive velocity, histological and immunohistochemical and the ultrastructure with transmission electron microscopy.Results:1.The result of frozen sections at each time point run thus: The experimental group: 24h(+++); 48h(++); 96h(++).The control group: 24h(++); 48h(+); 96h(—). The number of BMSCs homing to the injury site in experimental group was more than the control group apparently, it was the most abundant at 24 h, and then it decrease. 2. General observation:(1) the foot and leg of the operated side completely paralyzed, the toes were all together and the plantar prolapsed that lead the operated leg skid on the ground in each group. No foot ulcer and autophagy was found in the experimental group after operation.While there were four rats were found foot ulcers two weeks postoperation in the control group, these ulcers heal completely within two weeks, there was also no autophagy.(2) the recovery of autonomic activities: In the experimental group the rats started to recover the autonomic activities from three weeks postoperation, while the control group was about five weeks.3. Morphology of the anastomotic site: the anastomotic site of the experimental group was less adhesion to the surrounding soft tissue than the control group, and also the diameter of the anastomotic site closer to nomal. The diameter of the anastomotic site in the control group was thinner than nomal, and also the number of the regenerate capillary was lesser than the experimental group.4. The sciatic functional index(SFI) : The SFI value of each group revealed varying degrees of improvement gradually, and also the SFI of the experimental group showed significantly better recovery than the control group in each time point(P < 0.05).5. Histopathological and immunohistochemical analysis:(1) the nerve fibers reached to the distal of interstice can be found in each group. The state of neurofibra arrange, density of regenerate nerve fibers in unit area, the swelling degree of axons in experimental group were superior to the control group.(2) the experimental group: S-100(+++);The control group: S-100(++). The hyperplasia of Schwann cells in experimental group was more than the control group.6. The electrophysiological examination:The motor nerve conductive velocity(MNCV) value of each group revealed varying degrees of improvement gradually, and also the MNCV of the experimental group showed significantly better recovery than the control group in each time point(P < 0.05).7. Observation of the ultrastructure with the transmission electron microscope: The morphological structure and the number of nerve fiber, the morphological structure and the diameter of the axons of the myelin sheath in experimental group were better than the control group in each high power field.Conclusion:The BMSCs transplanted via caudal vein can homing to the injured peripheral nerve, while local low dose X-ray radiation promoted the homing of BMSCs to injured peripheral nerve apparantly obviously, so that help to accelerate the regeneration of neural function, but the specific mechanism of this effection needs further study.