The Study On Spinal Cord Injury Repair By LIPUS Mediated IPS-NSCs

Objective:Spinal Cord Injury(SCI)is a severe spinal traumatic disease with many treatments,including surgery,medication and cell transplantation.Neural stem cell transplantation is the focus of current research due to its remarkable repair ability of spinal cord injury.Induced pluripotent stem cells(iPSCs)induced neural stem cells(iPS-NSCs)are a kind of pluripotent stem cells with self-renewal and multi-differentiation potential.However,stem cells have low survival rate in vivo and it is difficult to differentiate into neurons.Thus,how to promote the proliferation and differentiation of iPS-NSCs is the focus of many researchers.Previous studies have shown that low-intensity pulsed ultrasound(LIPUS)is a physical stimulation method that can promote proliferation and differentiation of multiple cells.In this study we aimed to determine the effect of LIPUS on iPS-NSCs and its role in the repair of spinal cord injury and ultimately obtain a new stem cell optimization strategy.Method:Experiment in vitro:CCK-8 was used to identify the optimal LIPUS parameter according to the change of the proliferation activity of iPS-NSCs and LIPUS-iPS-NSCs.The TUNEL and ELISA method was used to detect the change of apoptosis and the expression of neurotrophic factor of LIPUS-iPS-NSCs;Western blot and immunofluorescence was used to detect cell differentiation.Western blot was adopted to detect the change of signal molecules related to notch signaling pathway including Notch 1 and Hes1.Experiment in vivo:The model of contusion SCI was established with Impactor model-II spinal cord hitter.The rats were randomly divided into 4 groups:sham group,control group,iPS-NSCs and LIPUS-iPS-NSCs transplantation group.The treatment and cell transplantation was performed 7 days after spinal cord injury.The structure and NTFs expression changes of the spinal cord tissue were tested by HE,immunofluorescence staining and ELISA.BBB locomotor rating scale was implemented to evaluate the motor function of the hind limbs.Result:In this study,the LIPUS system was successfully constructed.iPS-NSCs stimulated with LIPUS in vitro could increase their proliferation activity,and the optimal stimulation parameters are 1MHz and 8V(69.3 mW/cm~2).No significant differences in the apoptotic rate of iPS-NSCs stimulated at the optimal parameters.LIPUS stimulation could promote the expression of BDNF and NGF of iPS-NSCs in the supernatant.LIPUS intervention activates Notch signaling pathway and promotes the expression of Notch1 and HES1 in iPS-NSCs.The immunofluorescent staining of spinal cord showed the cavity of the injured spinal cord was reduced,the sprouting of nerve fiber and axon regeneration was increased and meanwhile reactive astrocytes were decreased obviously in the LIPUS-iPS-NSCs transplantation group.ELISA test showed that the expression of BDNF and NGF in the SCI leison was highest in the LIPUS-iPS-NSCs transplantation group.The recovery of hindlimb motor function in the LIPUS-iPS-NSCs transplantation group was significantly better than other injury groups.Conclusion:In this study,the self-developed LIPUS system was used to stimulate iPS-NSCs.LIPUS stimulation can obviously promote the proliferation ability and improve the expression of BDNF and NGF without affecting apoptosis.LIPUS is able to stimulation promote the differentiation of neurons into neurons while inhibiting the differentiation of glial cells;LIPUS may enhance the proliferation activity of iPS-NSCs by activating Notch signaling pathway.After transplantation of LIPUS-optimized iPS-NSCs,the motor function of hind limbs in SCI rats was significantly improved,which reduced the formation of local glial scars,promoted axonal regeneration and NTFs expression.This study demonstrates that LIPUS is a safe,non-invasive physical intervention in vitro that can improve the effect of iPS-NSCs cells in spinal cord injury.This study proposes a new strategy to optimiz the seed cells for tissue engineering and provides a new idea for the repair of spinal cord injury.