Study On Immune Regulation Of Bone Marrow Mesenchymal Stem Cell-derived Exosomes In Preterm Infants With Brain Injury

?Objective?:To observe the effects of exosomes derived from bone marrow mesenchymal stem cells on interleukin-6(interleukin-6)and transforming growth factor-?(TGF-?)in preterm infants with brain injury.To investigate the possible mechanism of paracrine effect of bone marrow mesenchymal stem cells(BMSCs)in intervening brain injury in preterm infants.?Methods?:1.Rat bone marrow and femur bone marrow were taken.BMSCs were isolated and cultured by whole bone marrow adherence method.Stem cell morphology was observed under inverted fluorescence microscope;BMSC-derived exosomes were enriched by ultracentrifugation.Transmission electron microscopy was used to determine the size of the extracted exosomes and protein electrophoresis to determine the exosome-specific expression of protein molecules.2.The neonatal mice of 3 days old clean SD rats were randomly divided into four groups:CONTROL group,WMD+PBS transplantation group,WMD+SUPERNATANT transplantation group and WMD+EXO group,and the brain injury model for preterm infants was made(white matter).Damage,WMD).WMD+PBS transplantation group,WMD+SUPERNATANT transplantation group,and WMD+EXO transplantation group were subjected to sevoflurane inhalation anesthesia,and the left common carotid artery was ligated to inhale oxygen-nitrogen mixed gas having an oxygen concentration of 6% for 2 hours to produce preterm labor.White matter damage model.After the mice in the control group were anesthetized,only brain models of preterm infants were made and no other treatments were performed.In the WMD+PBStransplantation group,5ul of PBS solution was injected through the left ventricle after injection.The WMD+SUPERNATANT transplantation group was injected with 5ul of SUPERNATANT via the left ventricle after the model was established,and the WMD+EXO transplantation group was injected with 5ul of EXO by the left ventricle after modeling.The control group was not given intracranial injection.3.After the model was successfully made,except for the CONTROL group,the remaining experimental groups were stereotactically transplanted into the lateral ventricle respectively with PBS,SUPERNATANT,and EXO.The histopathological changes of the brain and immunohistochemistry were observed using hematoxylin-eosin(HE)staining.The number of IL-6 and TGF-? positive cells in the rat brain;After 1 day,2 days,3 days,5 days and 7 days of intervention respectively,enzyme-linked immunosorbent assay and RT-PCR were used to detect IL-6 in each group.And TGF-? secretion levels and mRNA expression levels.4.Statistical analysis: SPSS 16.0 statistical software was used for statistical analysis of experimental data.The data of normal distribution were expressed as mean±standard deviation((x|-) ± s).For the repeated measurement data in this study,the repeated measures analysis of variance was used for comparison and analysis,and the comparison between groups was performed using the BONFERRONI test.The t-test was used to compare the mean of the two samples.P<0.05 was statistically significant,and P<0.01 was statistically significant.?Result?:1.The third generation of bone marrow mesenchymal stem cells were successfully cultured and purified by the whole bone marrow adherence method.The third generation of cells was whirlpool-like growth.BMSCs-exo was enriched by ultracentrifugation and identified by transmission electron microscopy.Nm and Western-blot were used to detect the surface marker proteins CAL-1 and CD63 of exosomes.2.After 1 day of intervention,the results of HE staining showed that the control group and WMD + PBS transplantation group showed partial cell degeneration and lateral ventricle dilation in the white matter area of the neonatal rats,and the structuralvariation of the hippocampus was obvious;WMD+SUPERNATANT transplantation group and WMD+EXO transplantation group Mild cell edema and degeneration were found in white matter areas of neonatal rats,but the extent of dilatation of lateral ventricles was smaller in the other two groups,and the structural variation in the hippocampus was lighter.The degree of variation in the WMD+EXO transplantation group was the least.3.The results of immunohistochemistry showed that the number of IL-6 positive cells in the brains of sucking mice of the CONTROL group and the WMD+PBS transplantation group was significantly more than that of the WMD+SUPERNATANT transplantation group and the WMD+EXO transplantation group transplantation group(BONFERRONI test,P values < 0.05);Control group and WMD + PBS transplantation group of rat brain TGF-? positive cell number was significantly less than WMD + SUPERNATANT transplantation group and WMD + EXO transplantation group transplantation group(BONFERRONI test,P value <0.05).4.ELISA showed that after 1 day,2 days,3 days,5 days and 7 days of intervention,the level of IL-6 protein in the brains of suckling mice of the CONTROL and WMD+PBS transplantation group peaked on the fifth day of the experiment and then decreased.On the 7th day,the levels of IL-6 protein in the WMD+SUPERNATANT transplantation group and the WMD+EXO transplantation group were significantly lower than those in the CONTROL and WMD+PBS transplantation group,and the 5th day time.The expression of IL-6 protein in the WMD+SUPERNATANT transplantation group and the WMD+EXO transplantation group was the lowest,and the difference was statistically significant(BONFERRONI test,P<0.05).ELISA showed that after 1 day,2 days,3 days,5 days and 7 days of intervention,the levels of TGF-? protein in the brains of suckling mice in the CONTROL and WMD+PBS transplantation groups were the lowest on the 5th day of the experiment,followed by a slight increase.However,the levels of TGF-? protein in the WMD+SUPERNATANT transplantation group and WMD+EXO transplantation group were significantly higher than those in the control and WMD+PBS transplantation group at the 5th day after the start of the 7th day.The expression of TGF-? protein in theWMD+SUPERNATANT transplantation group and the WMD+EXO transplantation group was significantly higher than that of the CONTROL group(BONFERRONI test,P<0.05).?Conclusion?:This experiment successfully collected culture supernatants and enriched bone marrow mesenchymal stem cell-derived exosomes,and identified by transmission electron microscopy the morphology of murine bone marrow mesenchymal stem cells-derived exosomes in a round shape,with a diameter of 40 Weartn-blot was used to identify exon-specific surface marker molecules CAL-1 and CD63 at 60-80 nm.Mouse model of hypoxic-ischemic hypoxic treatment in 3 days old pups was consistent with the pathological changes of white matter damage in preterm infants.SUPERNATANT and BMSCs-EXO transplantation can effectively improve the morphology of injured cells in the neonatal rat brain and the degree of hippocampal variation.Among them,the EXO transplantation group has the most significant effect.It can improve the brain damage caused by hypoxia-ischemia and regulate the immune and inflammation mechanism in the brain,may be related to the exosome secretion of bone marrow mesenchymal stem cells through the paracrine mechanism,the extracellular secretion of cells carrying RNA regulating neuroinflammation,down regulation of inflammatory cytokines IL-6 and upregulation of inflammatory cytokines TGF-? expression,BMSCs-exosome It has a neuroimmunomodulatory effect,which in turn reduces the mechanisms involved in early inflammatory responses.