Study On The Therapeutic Effect And Mechanism Of Canine Mesenchymal Stem Cells On Spinal Cord Injury

Canine spinal cord injury(SCI)is a disease with a high clinical disability rate and conventional treatments have limited effects.Mesenchymal stem cell(MSC)therapy is an emerging therapy in the past ten years.Clinical trials have shown that it has a strong repairing effect on damaged tissues.However,there are few studies on canine MSC in the treatment of SCI which showed its therapeutic effect and mechanism of action uncertain.Therefore,this article has carried out the following research:1.Preliminary verification of canine MSC treatment of rats40 rats were randomly divided into a blank control group,a sham operation group,an SCI+adipose-derived mesenchymal stem cells(ADMSCs)group,an SCI+bone marrow mesenchymal stem cells(BMMSCs)group and SCI+phosphate buffer solution(PBS)group.Except for the blank control group,the laminectomy of rats in other 4groups was performed.SCI+ADMSCs group,SCI+BMMSCs group and SCI+PBS group were subjected to the modified Allens method to prepare rat SCI models.After modeling,SCI+ADMSCs group and SCI+BMMSCs group were injected with canine MSC at 1×10~6 cells/kg at 1,2,3,7,and 14 days.The SCI+PBS group was injected with the same amount of PBS solution.The remaining two groups will not be injected.BBB(Basso,Beattie and Bresnahan locomotor rating scale,BBB scale)scoring method was used to evaluate the recovery of hindlimb motor skills after MSC treatment in rats.ELISA and immunohistochemical methods were used to detect inflammation factor in plasma and injured spinal cord tissue.TUNEL would be used to detect apoptosis of spinal cord tissue.LFB staining method is to detect myelin content and immunofluorescence histochemical method was used to detect glial fibrillary acid protein(GFAP)content.The results showed that at day7,14,21,and28,the BBB scores of the rats in the SCI+ADMSCs group and SCI+BMMSCs group were significantly higher than those in the SCI+PBS group(P<0.01).Content of TNF-αat SCI+ADMSCs group and the SCI+BMMSCs group in plasma and tissues of rats was significantly lower than that in the SCI+PBS group(P<0.05).The apoptosis rate and spinal cord scar tissue content in the SCI+PBS group were significantly higher than those 4 groups(P<0.05)and the content of spinal cord myelin sheath was significantly lower than the other 4 groups(P<0.05).The results suggest that both canine ADMSC and BMMSC can promote the repair of SCI in rats,and its repair effect may be achieved by reducing inflammatory factors,reducing the rate of apoptosis,reducing scar tissue formation,and increasing or maintaining myelin content in spinal cord tissue.2.The effect of canine ADMSCs on the proliferation,migration and related gene expression of RSC96 cells under hypoxia and hypoglycemiaIn order to explore the mechanism of MSC increasing myelin content in spinal cord tissue,this article used rat Schwann cells(RSC96 cells)as the object,and established a hypoxic and hypoglycemic cell model to simulate the tissue microenvironment during SCI.The CCK-8 method,scratch test and RT-PCR method would be used to detect the effects of ADMSCs on the proliferation,migration and related gene expression of RSC96 cells with hypoxia and hypoglycemia.The results show that ADMSCs can significantly promote the proliferation and migration of RSC96cells with hypoxia and hypoglycemia,and up-regulate the m RNA expression levels of proliferation(Ngf,NT-3)、migration(Nrg-1)and apoptosis(Bcl-2,Bax)related genes and significantly increase the ratio of Bcl-2/Bax.The above results suggest that canine ADMSCs may increase the myelin content by promoting the proliferation,migration and anti-apoptosis of Schwann cells,which may promote spinal cord injury repair.