Study On The Effect Of Electrical Stimulation In Promoting Bone Marrow Stromal Cells Differentiating Into Nerve Cells

Peripheral nerve and spinal cord injuries are currently common clinicaldiseases in Orthopaedics, which have several characteristics, such as highincidence, seriously injured degree, bad repairing effects, high morbidity andhigh cost. To “reconstruct structure continuity and accelerate nerve growthspeed” has been internationally recognized as the key of nerve repairing.However, these problems has not been effectively resolved. Tissue engineeringtechnology with cell transplantation has brought hope for nerve regeneration,but the application is limited mostly due to lack of source and immune rejection.The bone marrow stromal cells have highly self updating ability andmultiplex differentiation potential. They are considered to be the ideal seed cellsof tissue engineering because they can be induced differentiating to neurons. Atpresent, the commonly used chemical inducing methods have toxic effects oncells, which can shorten cell survival time, lead to function decline afterinduction, and can not meet the demand of transplantation. Therefore, it isnecessary to find a safe and effective method for cell inducing differentiation. In recent years, in the research of treatments of nervous system diseases andinjuries, the use of functional electrical stimulation increases rapidly. researchersfound that pulsed direct current electrical fields can promote ADSCsdifferentiate to osteoblasts. In addition, it is reported that low pressure pulseelectric fields jointed with5-nitrogen can promote rats BMSCs differentiated tocardiocytes. These evidence indicated that electrical stimulation can probablyintervene cell differentiation process. But so far, there has no report on ES, thatinduced BMSCs differentiating into neurons. So it is important to prove itseffectiveness and find out feasible method.Our subject plans to use a kind of physical way, electrical stimulation, toinduce the process of differentiation on BMSCs. Firstly, we plan to screen anddetermine the appropriate parameters. Then we are goin to research from severalaspects, such as cells form, cell proliferation capacity, cell apoptosis situationand cell differentiation, in order to provide new method for nerve repairing.Part one：Separation, cultivation and identification of the rat BMSCsObjectives Separate, cultivate and purify the rat BMSCs, identify the cells bymorphological observation, flow cytometry technology and osteoblastdifferentiation experiments.Methods By using the whole bone marrow culture method to obtain and cultureBMSCs of SD rats, observing cell morphology change by inverted microscope,testing different algebra cell proliferation ability by CCK-8, testing expressionof CD29, CD90, CD45by flow cytometry technology, inducing the P3BMSCsdifferentiating to osteoblast by bone induction medium in order to prove the differentiation ability.Results By using the whole bone marrow culture method, we succeed inculturing BMSCs of good growth state.CCK-8testing result showed that thecultured cells had good proliferation ability. The flow cytometry technologyresult showed that most cells were CD29and CD90positive, CD45negative andthe ratio is96.89%. The osteoblast differentiation experiments indicated that theinduced cells can express alizarin red and ALP.Conclusion Through cell morphology observation, cell proliferation capacitytesting, flow cytometry technology identification and osteoblast differentiationexperiments, we proved that the whole bone marrow culture method can obtainBMSCs of good activity, highly self updating ability and multiplexdifferentiation potential, which can provide cell sources for the follow-upstudies.Part two：Effect of electrical stimulation on BMSCs induced differectiationprocessObjectives By using the electrical stimulation induced BMSCs differentiatinginto neurons, we plan to explore the optimal stimulating parameters andpreliminary clarify the effect of electrical stimulation on BMSCs induceddifferentiation to neurons and provide new way for seed cells transplantationtherapy on nerve repairing.Methods (1) By applying direct current stimulation, we choose different timeand different voltage to deal with the cultured BMSCs.CCK-8tested cellproliferation capacity change, flow cytometry technology tested cell apoptosis situation and then find out the optimal electrical stimulation parameters.(2) Usethe optimal electrical stimulation parameters to the induced differentiationprocess research. Set up ES group, Chemical group, Complex group and Controlgroup respectively. Use MAP-2immune fluorescence technology to identify theinduced differentiation situation and calculate differentiation ratio.Results (1) The time screening experiment result indicated that, when setvoltage to3V, after applying stimulating time as30min/d(group B),the cellsform and number were obviously superior to other groups; the cell proliferationcapacity was improved when compared with other groups and the difference hasstatistics significance; the cell apoptosis experiment has not found the cellapoptosis situation increased. Then the voltage screening experiment find thesame result as above. Therefore, we confirm3V、30min/d as the optimalelectrical stimulation parameters.(2) Use the conventional chemical inducedmethod to induce BMSCs differentiate into neurons as the positive control group,through the comparison of the induction rate and immunofluorescence stainingresults, we found that both ES group and Chemical group can induce BMSCsdifferentiating into neurons and the difference has no statistics significance（P＞0.05）; the induction rate of the Complex group is much higher than the ESgroup and Chemical group and the difference has statistics significance（P＜0.05）.However, in Chemical group and Complex group, after induction, thecells began to rupturing and crumbling, which has not been seen in the ESgroup.Conclusion By screening different time and different voltage, we confirm3V、30min/d、 continuously for2days as the optimal electrical stimulationparameters and use these parameters in the induced differentiation experiment. The results indicated that electrical stimulation not only can achieve the sameinduced effect with chemical inducer, but also has less toxic effect. Our subjectprovided experimental evidence for ES and stem cell therapy and provided anew way for the establishment of the seed cells of tissue engineering.