| Spinal cord injury (SCI) accounted for about0.2to0.5%of the whole bodytrauma, and its treatment is a worldwide problem in clinical medicine. According toincomplete statistics, the number of SCI has reached millions in China, and stillincreasing at an annual number of120000. With the deepening of the study of stemcells for spinal cord injury, neural stem cells applied to treat spinal cord injury havemade some progresses. But efficiency of the differentiation of spinal cord-derivedneural stem cells into neurons is very low and it is difficult for them to have someimportant functions, which is different to neural stem cells in the brain. This hasalways been the difficulty of the neural stem cell research. Recently, the researchesconcerning the microenvironment of the migration, differentiation and regulation ofneural stem cells have gradually become a hot spot. It has found that not because thespinal cord-derived neural stem cells lacking their potential to differentiate intoneurons, but lacking their specific microenvironment. A variety of biological methods,by changing the internal and external environment to promote its spinal cord-derivedneural stem cells differentiating into neurons, proved to be less effective, harmful oreven have the potential to induce tumor. Therefore a brand new safer and convenientway is mandated.There have been a long history for human beings applying current as a treatmentto some diseases, mostly of them are empirical, which could cause great damages tothe patient. With the progress of science and technology, the study of humanbioelectricity gradually deepened. It has been found that the bioelectric phenomenawidely exist in all kinds of organisms, and the effect of these phenomena is widelyinvolved in development of embryo and repair of tissue after injury. And it hasconfirmed that the migration and proliferation of nerve cells in the process of humandevelopment and even the transport of tumor cells have close relationship with thephenomena.Application of external electrical stimulation can contribute to the repair of nervedamage and healing of skin wound. Some in vivo studies have found that currentstimulation of nerve tracts can cause proliferation, migration and differentiation ofendogenous neural stem cells/nerve premise cells in central nervous system. But thiseffect of external current stimulation has a big difference with the endogenous currentfield effect of natural organism. Endogenous current field effect is mainly due to theuneven distribution of charged particles inside and outside the cells, tissues and organs. This endogenous bioelectricity effect in the study of microenvironment of theneural stem cells activation and regulation has gradually become a hot research.Therefore, the study of biomimetic weak current field has captured more attentionfrom scholars both at home and abroad. Its research is of great significance.Based on our previous research in biological neural microelectrode repairingnerve injury, in this study we will build a biomimetic weak current field cell culturesystem in vitro, furthest simulating the internal environment, fully considering thecharacteristic physical factors of biological current field, and then to observe theeffect of biomimetic weak current field in differentiation and migration of spinalcord-derived neural stem cells. We will further explore the concerned regulatorymechanism to provide theoretical basis and new treatment strategies for applyingspinal cord-derived stem cells to treat spinal cord injury.Part one：Separation, cultivation and identification of the mouse spinalcord-derived NSCsObjective: Separate,cultivate and purify the mouse spinal cord-derived NSCs,identify the stem cells by morphological observation, immunofluorescence technologyand multi-lineage differentiation experiments.Methods: By using the suspension culture method to obtain and culture spinalcord-derived NSCs of E13.5d mouse, observing cell morphology change by invertedmicroscope, testing different algebra cell proliferation ability by CCK-8testingexpression of Nestin and Sox2by immunofluorescence technology, inducing the P4spinal cord-derived NSCs differentiating to mutilineage differentiation by naturaldifferentiation method in order to prove the differentiation ability.Results: Serum free medium suspension culture method has been appliedsuccessfully in seperating spinal cord-derived NSCs. CCK-8absorbance good experimental detection cell proliferation activity. Using immunofluorescence chemicaltechnology, mark the cell surface markers found cells high expression of Nestin andSox2, after image fusion with DAPI nuclear dye markers,showing highly uniform.Further differentiation showed that cells can express both Tuj1and GFAP marker.differentiation potential is good.Conclusion: Serum-free suspension culture method, can be successfully used incultivating NSCs. After proliferation activity, immunology and apoptosis detection, itshows that the cell vitality is well,actived differentiation potential. Part two：NSCs proliferation and differentiation process in biomimetic weakelectric fieldObjective: To culture spinal cord-derived NSCs continuously in the weakcurrent field, exploring the parameters of its proliferation and induction into neurons,preliminarily clarifying the effect of weak current field on spinal cord-derived NSCsinduced differentiation into neurons. We hope to provide new experimental data forbiophysical methods to be applied in reconstructing and repairing of spinal cordinjury.Methods: Build biomimetic weak field workstation in cells chamber, currentintensity (A:20mV/mm, B:60mV/mm, C:100mV/mm, D:140mV/mm, E: blankcontrol) and the liquid level height (A:1.5x2,400um, B:1.5x4,800um,C:1.5x6,1200um,D:1.5x8,1600um, E:normal high liquid (2500um)).After intervention,useCCK-8regent to detect change of cell proliferation,flow cytometry technology to testthe effects of electrical stimulation of cell apoptosis,screening the most suitablemicroenvironment of weak current field specific parameters.(2) According to selectedsuitable weak current field data on differentiation microenvironment, set up electricalstimulation induced group respectively, natural differentiation in the control group.Use GFAP and Tuj1immunofluorescence staining to identify situation of celldifferentiation, and to calculate the differentiation ratio.Results:(1) Weak current field screening experimental results show that whenthe intensity setting is60mv/mm, the liquid level height of800um, after three timepoints stimulation, cells form and quantity were significantly better than the rest of thegroup. Cell proliferation ability increased significantly than the rest of the group, andthe difference is statistically significant. Apoptosis detection found that the parametersof electrical stimulation were not significantly increased. Screening experiment resultsshow that when the intensity of60mv/mm, the liquid level height of800um, afterstimulation, cells form and quantity is better than the other groups, cell proliferationability improved obviously, and the difference is statistically significant. Apoptosisdetection show that the parameters of electrical stimulation not significantly increasedcell apoptosis. To determine the intensity of60mv/mm, the liquid level height of800um is suitable for electrical stimulation and environmental parameters.(2) Accordingto the experimental results, in cell culture chamber, the application of the conventionalnatural derivation by contrast, through the comparison of induction mouse andimmunofluorescence staining results, found that number of induction of NSCs differentiation into neurons was statistically significant differences (p <0.05) in theweak current group.Conclusion: The experimental results show that determining the intensity of60mv/mm, the liquid level height of800um for electric field parameters is suitable.Application of parameters in the NSCs differentiation of neurons show that toxicityand injury effect of electrical stimulation is small, it can be more safe and effective forNSCs differentiation. This experiment could provide new experimental data forbiophysical methods to be applied in reconstructing and repairing of spinal cordinjury. |
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