Experimental Study Of Mesenchymal Stem Cell Transplantation In The Treatment Of Stroke And Post-stroke Infection

Stroke with the high incidence of disability and mortality,and lack of effective treatment,is the current social and medical problems to be solved.At present,the main treatments for stroke were intravenous thrombolysis,interventional thrombolysis,antiplatelet and anticoagulation therapy,and neuroprotective therapy,but all of them have certain limitations.Mesenchymal stem cell,especially adult mesenchymal stem cell such as bone marrow mesenchymal stem cell(BMSC),adipose mesenchymal stem cell(ADSC),umbilical cord mesenchymal stem cell(ucMSC),was good seed cell used in the treatment of stroke by transplantation,its'main mechanisms were as mainly involved as below.The regulation of immune inflammation,the secretion of nutrients or growth factors,promotion of nerve regeneration and angiogenesis,the possible structure and function integration.However,in the study,there are still many problems affecting the therapeutic effect of MSC transplantationFirst,the mobilization of stem cells involved in brain functional repair after stroke.Previous studies have showin that no natter transplantation or in-situ mobilization of MSC can directly migrated into brain lesion areas,and SDF-1/CXCR4 plays an important role in the process of mobilization of MSC.However,it was found that the low homing efficiency of mesenchymal stem cells affected the therapeutic effect.OSM has the functions of inhibiting inflammatory response and excitatory nerve injury,protecting neurons and promoting axon elongation and is closely related to the expression of SDF-1.However,it is not clear whether OSM has an effect on migration of stem cells involved in brain functional repair after stroke Therefore,in the study,we explored whether OSM can enhance the homing efficiency and therapeutic effect of BMSC.BMSC were used in this part of the experiment,which was based on a lot of previous work of our teamSecond,secondary infection after stroke and the effect of stem cell therapy on secondary infection after stroke.In general,ischemic stroke can lead to the activation of the hypothalamic-pituitary-adrenal axis,resulting in excessive infiltration of lymphocytes(such as NK cell)into the brain,aggravating brain injury.Meanwhile,the distribution of peripheral immune system is insufficient,which leads to the decline of body immunity and induce post-stroke infection easily.Many studies have shown that mesenchymal stem cells have regulatory effects on lymphocytes such as T cells,NK cells,dendritic cells,macrophages,all these studies suggested the feasibility of-MSC in inhibiting the infiltration of CD8+T cells and NK cells.However,the potential problem is whether MSC transplantation can lead to or aggravate post-stroke infection by inhibiting peripheral immune function.Therefore,in the the study,we explore the eff-ect of MSC transplantation on CD8+T cells and NK cells in the ischemic stroke model,as well as the relationship with post-stroke infection and the potential mechanism.Considering the facilitation of clinical research on the source of stem cells,ucMSC was adopted in this part of the study.Part 1:The effect and mechanism of OSM on intracerebral migration efficiency of transplanted BMSC and prognosis of ischemic strokeObjective:To explore whether oncostatin M(OSM)combined with bone marrow mesenchymal stem cell(BMSC)treatment can improve BMSCs migration,as well as the prognosis of ischemic stroke.Methods:BMSC and astrocytes were treated with OSM in vitro,and the expression of SDF-1 was detected by RT-PCR,ELISA,immunofluorescence.The effect of OSM on BMSC migration was analyzed by Tranwell culture chamber.An 4-0 suture was inserted from the left external carotid artery into the middle cerebral artery in rats to set up ischemic stroke model.To analyze OSM secretion,total protein was collected from ischemic stroke rat brains after 0-72h for Western blotting detection.Ventricle injection of OSM combined with caudal vein graft of-BMSC(3*106)was performed in ischemic stroke rats.After 72 hours,production of SDF-1,IL-1?,VEGF,IL-6 and grafted BMSC in the lesion areas of cortex were detected,while nervre function score were evaluated too.Results:OSM can enhance the expression of SDF-1(P<0.01),VEGF(P<0.01)and MMP-2(P<0.01)of BMSC.OSM can promote the secretion of SDF-1,which promotes the migration of BMSC(P<0.01).The combination treatment of OSM and BMSC in MCAO rats increased the migration efficiency of BMSC in the brain(P<0.05),which not only improved neurofunctional recovery significantly(P<0.05),but also reduced the expression of inflammatory mediators and promoted the secretion of nutrition factors.Conclusion:OSM can promote the migration of BMSC by up-regulating the expression of SDF-1 in BMSC and astrocyte.OSM combined with BMSC in the treatment of ischemic stroke can improve the efficiency of BMSC transplantation and promote the recovery of neurological functionPart 2:The effect and mechanism of ucMSC on post-stroke infectionObjective:To explore the effect of ucMSC transplantation on immune response,the relationship between ucMSC transplantation and post-stroke infection,and the relevant mechanism.Methods:1 The mice underwent ischemic stroke or post-stroke infection were subjected to ucMSC,then assessed the area of brain damage by methylene blue staining and neurological motor function by mNSS scoring system,detected the the distribution of CD8+T cells and NK cells in the brain,peripheral blood and spleen by immunohistochemistry,immunofluorescence and flow analysis.Histopathology and bacterial culture was performed to analyse the injury and infection of each tissue and organ.Meanwhile,the blood routine,blood biochemistry,the content of inflammatory factors in peripheral blood,and the number and function of platelets were measured3 Platelets were stimulated with different concentrations of LPS or co-cultured with ucMSC in vitro,autophagy and apoptosis were analyzed by WB.Escherichia coli were co-cultured with platelets and ucMSC,and the concentration of escherichia coli was examined at different times to detect the proliferation efficiency of the bacteria.Results:1 mNSS scores and the area of brain injury in the ucMSC treatment group decreased significantly(P<0.05).ucMSC treatment significantly reduced the infiltration of CD8+T cells and NK cells in the brain(P<0.05).There were no obvious signs of infection in the organs of mice after stroke model and stem cell therapy.2 ucMSC treatment alleviated inflammatory cells infiltrating in the lung,brain,and liver and caused cell and tissue damage.reduced the level biochemical indicators aspartate aminotransferase(AST),alanine aminotransferase(ALT),creatine kinase(CK),lactate dehydrogenase(LDH)and the expression of inflammatory cytokines(TNF-a,IL-6),while enhance the expression of anti-inflammatory factor IL-10(P<0.05).It's worth noting that the ucMSC transplantation can significantly reduce the decline of platelet number(P<0.05),maintain the level of platelet autophagy and the generation of ATP,and inhibit the apoptosis of platelets(subsequent experiments were further developed from this point).3 Western blotting analyses revealed that LC3-? and Beclin-1 levels were decreased,whereas that of p62 was increased,in platelets exposed to LPS;however,ucMSC treatment abolished these effects.Treatment with ucMSCs also reversed the upregulation of cleaved caspase-3 and downregulation of Bcl-2 and Bcl-xL induced by LPS stimulation.TEM analysis ucMSC inhibits the reduction of platelet autophagosomes and mitochondrial damage.Bacterial culture showed that ucMSC enhanced the antibacterial ability of platelets(P<0.05).Conclusions:1 ucMSC can alleviate brain injury by inhibiting the infiltration of CD8 T cells and NK cells in the brain.ucMSC have certain immunosuppressive effects,but do not lead to post-stroke infection.2 ucMSC can inhibit the apoptosis and maintain the function of platelets,while enhance the antibacterial ability of platelets to inhibit the progression of post-stroke infection.