Experimental Study On Peripheral Nerve Injury By Different Ways Transplantation Of MSCs With Small Gap Anastomosis Neurorrhaphy

Part1The separation, culture and identification of Bone marrow mesenchymal stem cellsObjective:To master the method for the isolation,culture and amplification of rat bone marrow mesenchymal stem cells(BMSCs) in vitro and explore their biological characteristics.Methods:BMSCs were collected from degermed femurs, and tibias of SD rat by flushing the shaft with L-DMEM using a syringe. Cells were disaggregated by gentle pipetting several times and plated in culture flask and re-fed every2-3days(L-DMEM with10%FBS).When90%fusion, cells were digested with trypsogen2-3min and passaged. After successive isolation, purification, subculture and proliferation, the morphology was observed with phase contrast microscope. The growth curve of P2,P4,P6were drawn by cell counting. The proliferation and morphology of BMSCs was observed with inverted microscope constantly. Cells were identified by Flow Cytometry.Results:The components of primarily cultured BMSCs were very complex. The marrow cells were round in the beginning and a few cells adhered to flask within first24h, which were in irregular shape such as fusiform, polygon and so on. The first2to5days the proliferation of cells were slowly. About6-9days later, cells might overgrow the bottom of culture flask. The cells arranged regularly as a whirlpool. Cells could spread the full flask bottom for12or15days. Generated cells stuck on the wall more quickly than primary cells. The cell morphology was more uniform and all the cells arranged more regularly after generated by4or5times. The growth curve of P2,P4,P6were quite similar and the cells biological character kept stable. The result of growth curve showed that cell growth phase was composed of latency phase (1-2days),logarithmic growth phase (3-5days) and platform phase (6-7days).The5th generation BMSCs were identified by flow cytometry:CD90(98.9%), CD29(93.7%) positive, CD34(0.8%), CD45(1.3%) negative.Conclusion: In this part, a simple new method which was used for the isolation, purification and cultivation in vitro of BMSCs from SD rat bone marrow by total marrow culture associating with adhering to flash has been established. The BMSCs could proliferate immediately and keep their biological character stable in vitro. Part2Experimental study on peripheral nerve injury by different ways transplantation of MSCs with small gap anastomosis neurorrhaphyObjective:To observe the therapeutic efficacy of peripheral nerve injury, through different ways transplantation of MSCs with small gap anastomosis neurorrhaphy. To find an effective method for the treatment of peripheral nerve injury.Methods s Rat models of peripheral nerve injury were established by small gap anastomosis neurorrhaphy.36SD rats with weight about250g were divided into3groups randomly, the control group(CG),the intravenous injection group(VG) and the intramuscular injection group(GG) with12rats in every group. The rats in the VG and GG group were transplanted by BMSCs labeled with BrdU,l week later.All animals of3groups were monitored for changes in their appearance and locomotion activities after surgery. Their posture and gait were recorded regularly with the aid of photographs for each rat. Evaluations of electrophysiological function, the sciatic functional index(SFI),the change of histochemistry and immunohistochemistry, the recovery rate of gastrocnemius wet weight, and the microscopy ultrastructure.Results:1. The incidence of foot ulcer in experimental groups was lower than the control group.2. SFI of every groups were gradually improved4weeks after surgery, the differences of SFI between every groups was statistical significance (P<0.05),8weeks after surgery, the differences of SFI between CG and VG, CG and GG was significant deviation (P<0.01).,the differences of SFI between VG and GG was statistical significance (P<0.05),12weeks after surgery, the differences of SFI among every groups was significant deviation (P<0.01).3.The differences of motor nerve conduction velocity among three groups was significant deviation (P<0.01).4. After operation the gastrocnemius muscle atrophy occured in all rats, however the degree of atrophy in GG was lightest.4weeks, and12weeks after surgery, the differences of the recovery rate of gastrocnemius wet weight between CG and VG without statistical significance (P=0.255, P=0.329),while the differences between CG and GG, VG and GG were statistical significance (P<0.05).5.The shape and distribution of the nerve fibers in the experimental groups were better than control group, meanwhile with comparison GG was better than VG. BMSCs labeled with BrdU were foud in nerve stump and cornu anterius medullae spinalis in the experimental groups, however the cells were not find in the control group.6.The layers of myelin sheath were arranged in concentric circles, compacted and regular and Schwann cell hyperplasia was obvious in GG group, without demyelination and axonal necrosis. While the layers of myelin sheath were arranged irregularly and sparsely, and the myelin sheath was obviously tortuous and blurry in CG group, and demyelination and axonal necrosis could be observed. The layers of myelin sheath arranged irregularly in VG., but Schwann cell hyperplasia was obvious.Conclusion:1BMSCs can home to damaged tissues after transplantation via intravenous injection or intramuscular injection in vivo.2Transplantation of bone marrow mesenchymal stem cells can promote nerve regeneration and functional recovery via intravenous injection or intramuscular injection.And the therapeutic effect of intramuscular injection is better than intravenous injection.3Transplanting bone marrow mesenchymal stem cells via intramuscular injection can delay the denervated muscle atrophy.