| Objectives: To explore the effects of mild hypothermia on differentiation property of transplanted temperature-sensitive umbilical cord mesenchymal stem cells after traumatic brain injury in the perspective of physical and chemical microenvironment.Methods:1 The establishment and cultivation of temperature-sensitive umbilical cord mesenchymal stem cells: Umbilical cord mesenchymal stem cells were isolated from umbilical cord of the newborn, while the temperature-sensitive umbilical cord mesenchymal stem cells(ts UC) were obtained through the transfection of ts-SV40 LT virus, and then be cultivated under environment of mild hypothermia or normal temperature.2 The fabrication of two-dimensional culture medium of stem cells mimicking brain tissue elastic modulus: Acrylamide and bis-acrylamide of different concentration were applied to synthesize polyacrylamide hydrogel of different elasticity modulus. The biomechanical testing machine was applied to stretch and test the elastic modulus of the polyacrylamide hydrogel, and the gels mimicking the rigidity of normal bran tissue and the brain tissue subjected to traumatic brain injury(approximate to muscle). According to the corresponding concentration ratio, the two-dimensional matrix simulating elastic modulus of brain tissue and muscle tissue were prepared.3 The influence of mild hypothermia on temperature-sensitive umbilical cord mesenchymal stem cells under the physical microenvironment of brain tissue after traumatic brain injury: There were three experiments in this study, including: Control, UC+NT+0.5k Pa, ts UC+MHT+0.5k Pa, UC+NT+8.0k Pa, ts UC+MHT+8.0k Pa. For the control group, umbilical cord mesenchymal stem cells onto a glass slide were routinely cultivated, and the rest four groups were sorted according to the elastic modulus of matrix, the cell type and cultivating temperature. Cell growth and morphological changes in each group were dynamically observed. Seven days later, cell immunofluorescence were implemented, with the differentiation level of each group estimated and the axon length of the differentiated neurons calculated by using Image J software.4 The influence of brain tissue extracts of injured brain treated by mild hypothermia on umbilical cord mesenchymal stem cells: Rat models of traumatic brain injury(TBI) were made by electric cortical contusion impactor combined with systemic mild hypothermia(MHT)(33℃) for 4 hours. After that, polyacrylamide gels mimicking the elastic modulus of brain were manufactured. Human UC were isolated and cultured on the gels, adding brain tissue extracts from rats of Sham(Sham group), TBI(TBI group), or TBI combined with MHT(TBI+MHT group) into culture medium. After 24 hours, the apoptosis level of UC was checked, and the medium were changed with normal one. Seven days later, cell immunofluorescence were implemented, with the differentiation level of each group estimated.5 The neural restoration of TBI brain after the transplantation of temperature-sensitive umbilical cord mesenchymal stem cells combined with mild hypothermia: Rat models of TBI were made and randomly divided into four groups: brain injury control group(TBI), umbilical cord mesenchymal stem cell transplantation group(UC), mild hypothermia combined with umbilical cord mesenchymal stem cell transplantation(MHT+UC), mild hypothermia combined with temperature-sensitive umbilical cord mesenchymal stem cell transplantation group(MHT+ts UC). Stem cells marked with Brd U were injected into the brain tissue around the focal injury by using stereotactic technology. Rats in the MHT+UC group and the MHT+ts UC group were immediately given hypothermia treatment after the establishment of TBI models, with after and stem cell transplantation was not carried out until the temperature dropped to 33℃. Three days later, brain water content was measured according to wet and dry weight. Neurological deficit score was evaluated at the time points of 24 hours, three days and seven days. Seven days later, the brain injury volume was calculated, and cell immunofluorescence were implemented to estimate the living condition of transplanting cells.Results:1 The cultivation and identification outcome of umbilical cord mesenchymal stem cells: The flow cytometry assay demonstrated that the expression of cell surface antigen conforms to the characteristics of umbilical cord mesenchymal stem cells. The purity of umbilical cord mesenchymal stem cells is 97.91%, and the growth, proliferation activity is exuberant.2 The cell cultivating matrix based on polyacrylamide hydrogels: Through the measurement and screening by biomechanical testing machine, polyacrylamide gels mimicking the rigidity of normal bran tissue and the brain tissue subjected to traumatic brain injury were obtained. The elastic modulus of PA gels was 0.5±0.03 k Pa and 8.0±0.11 k Pa.3 Morphological changes of stem cells on polyacrylamide hydrogels of different elastic modulus: A small amount of cells resembling the neurons were presented in group UC+MHT+0.5k Pa and ts UC+MHT+0.5k Pa, with small soma and long, slender protuberances. While the stem cells in group UC+MHT+8.0k Pa and ts UC+MHT+8.0k Pa had a performance of long spindles, like the muscular cells. Cells in control group had no transformation during the experiment period.4 The cell immunofluorescence on polyacrylamide hydrogels of different elastic modulus: Cell immunofluorescence statistics demonstrated that a small amount of cells in group UC+NT+0.5k Pa and ts UC+MHT+0.5k Pa differentiated into neurons with positive expression. The neuron differentiation rate and axon length were significantly higher than that of the other three groups which have no positive expression(*P<0.01). However, there was no obvious difference between group UC+NT+0.5k Pa and ts UC+MHT+0.5 k Pa(#P>0.05).5 The apoptosis level and cell immunofluorescence of stem cells under the intervention of different brain tissue extraction: According to the results, there were more apoptotic cells in UC+TBI group than in the UC+Sham group(*P<0.01). However, UC+TBI+MHT group significantly reversed the apoptosis of cells in TBI group(*P<0.01). Cell immunofluorescence results demonstrated that neuronal differentiation rate of UCMSCs in TBI+MHT group were obviously lower than that of TBI group(*P<0.01).6 Brain edema degree and neurological deficit scores of rats: Three days after TBI, the edema degree relieved in group TBI+ts UC+MHT, there were more water content in TBI+PBS group and TBI+ts UC group(*P<0.05,#P<0.05). Three days after TBI, the neurological deficit scores of rats in TBI+PBS group, TBI+ts UC group and TBI+ts UC+MHT group were lower than that of the first day(aP<0.05). Seven days after TBI, the neurological deficit scores of rats in TBI+PBS group, TBI+ts UC group and TBI+ts UC+MHT group were lower than that of the first day(bP<0.05). and the neurological deficit scores of rats in TBI+ts UC+MHT group was lower than that of TBI+PBS group and TBI+ts UC group as well(cP<0.05,dP<0.05).7 The calculation of injury volume of rats: The injury volume of rats in TBI+ts UC+MHT group was obviously small than that of TBI+PBS group, TBI+ts UC group(*P<0.05,#P<0.05).8 Cell immunofluorescence of rat brain tissue: the results demonstrated that there were more living transplanted stem cells in MHT+ts UC group, compared with TBI+ts UC+MHT group(*P<0.05).Conclusions:1 The differentiation property of temperature-sensitive umbilical cord mesenchymal stem cells under mild hypothermia is similar with normal umbilical cord mesenchymal stem cells. Temperature-sensitive umbilical cord mesenchymal stem cells can differentiate into neurons in the environment mimicking the elastic modulus of brain tissue under mild hypothermia, but can not differentiate into neurons in the environment mimicking the elastic modulus of TBI brain tissue.2 Mild hypothermia can improve the high physical microenvironment of TBI brain tissue, and can improve the survival rate and differentiation ability of transplanted temperature-sensitive umbilical cord mesenchymal stem cells, which eventually promote the neural functional recovery.3 Mild hypothermia can improve the chemical microenvironment of brain tissue after TBI. As a result, the apoptosis of transplanted umbilical cord mesenchymal stem cells can be decreased and the differentiation capacity partly restored. |
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