| Purpose: to study the differentiation and neural function structure protection of engrafted peripheral blood-derived mesenchymal stem cells(peripheral blood-derived mesenchymal stem cells,PB-MSCs) in rats after SCI. to evaluate application value of PB-MSCs as an alternative sources of mesenchymal stem cells(mesenchymal stem cells,MSCs) used in the treatment after SCI.Methods: 1 The 8-week old SD rats were performed subcutaneous injection of recombinant human granulocyte colony-stimulating factor(granulocyte colony stimulating factor,G-CSF) at 100 mg/kg/day for five days. Then 5~8 ml peripheral blood was collected from the left ventricle of each rat, from which the peripheral blood mononuclear cells(peripheral blood mononuclear cells,PBMNCs) were isolated through density gradient centrifugation and then fibroblast-like cells were obtained by adherent culture. Therefore, the phenotype of the cells from passage two(P2) was analyzed by flow cytometry and immunocytochemical staining. 2 Take the P2 generation of PB-MSCs via PKH26 fluorescent tags standby continue to develop to P3 generation after transplantation. 3 Adult healthy SD rats(N=66) were randomly divided into model group(N=15), the PB-MSCs transplantation group(N=21) and the PBS group(N=15) after tablishment of SCI model with an impactor. Molding success after 30 min PB- MSCs transplantation group with micro syringe 10 ?l PBMSCs suspension(2×104/?l) slowly injected into the spinal cord parenchyma, PBS group injected equal amount of PBS as a control. Another 15 SD rats only remove the lamina, no damage to the spinal cord were as sham operation group. 4The behavior and limb movement function were evaluated weekly via BBB scoring and Ravlin inclined plate experiment to assess the change of movement function in rats after injury. 52 weeks, 4 weeks, 8 weeks after taking damage area of spinal cord tissue paraffin section of HE staining to observe the histopathological changes;Immunohistochemistry of glial fibrillary acidic protein(GFAP), myelin basic protein(MBP), neurofilament protein(NF-200), microtubule associated protein-2(MAP-2) were analyzed to evaluate the glial scar formation, myelin and axon regeneration. 6Immunofluorescence technique to detect PB-MSCs transplantation group applications PKH26 positive PB-MSCs in injury of spinal cord tissue engraftment and survival, and dual fluorescence detection PKH26+GFAP+, respectively PKH26+MBP+and PKH26+ Neu N+ double positive cells, to study the PB-MSCs into glial cells, oligodendrocytes and neurons of differentiation.Result:1Fibroblast-like cells colonies obviously formed 9 days later and the cells could grow to approximately 80% confluence about 18 days later under primary culture; The passage PBMSCs possessed high adherent abilities, showing a uniform spindle growth pattern; 2 The results of FCM and immunocytochemistry suggested the enriched PBMSCs strongly expressed CD90, CD44, CD29, CD73 and CD105, but failed to express CD45, CD11 b, CD79 a, CD34 and HLA-DR. 3Postoperative 1w and 2w BBB score and Ravlin inclined plate experiment was no statistically significant difference between the model group, the PBS group and PB-MSCs transplantation group( P?0.05),from 3w after surgery, PB-MSCs transplantation group showed a higher BBB scoring and a lager angle(P<0.01), no statistical difference between model group and PBS group(P?0.05). 4H&E staining results: there were unclear boundary between gray and white matter, severe disintegration of neuron-like cells, cavity formation and gliosis at 2 w after SCI of the model group, the PBS group and PB-MSCs transplantation group. At 4 w afteroperation, there were less significant gliosis after PBMSCs injection than that in the other two groups. At 8 w, significant gliosis aggregated to glial scars in the model and PBS groups, while there was no obvious change and less cavities in the PB-MSCs transplantation group. 5Immunohistochemical results: PB-MSCs transplantation group of GFAP expression is lower than the model group and PBS group(P<0.01), and a higher expression of MBP, NF-200 and MAP-2(P<0.01). 6PB-MSCs transplantation group of spinal cord tissue after transplantation of frozen section can be observed when 2 w, 4 w and 8 w damage area PB-MSCs are alive, and saw PKH26+GFAP+, PKH26+MBP+and PKH26+Neu N+ double fluorescence positive cells.Conclusion: PB-MSCs transplantation can significantly improve the SCI rats hind limbs motor function recovery; Orthotopic transplantation of PB-MSCs in rats in vivo engraftment and survival at least 8 weeks, and to be able to differentiate into neurons, astrocytes and oligodendrocytes; at the same time, PB-MSCs transplantation can reduce the formation of glial scar, protective myelin sheath, promote the regeneration of the neuron axons. The result provides the PB-MSCs transplantation in the treatment of SCI clinical research evidence before the experiment, and is expected to become the ideal alternative resources MSCs for the treatment of SCI... |
PDF Full Text Request