| Objective:By constructing a transient middle cerebral artery ischemia-reperfusion(MCAO)model in rats,we investigated the expression levels of Zeb2 gene in bone marrow mesenchymal stem cells(BMSCs)and exosomes after ischemic brain injury in Sprague-Dawley(SD)rats.To explore the significance of Zeb2 in BMSCs-derived exosomes in ischemic brain injury in rats.Methods:250-350 gram SD male rats were randomly divided into model(MCAO)group and sham(Sham)group.In MCAO group,the right internal carotid artery was ligated to establish the transient MCAO model in SD rats.In sham group,the internal carotid artery was ligated without blocking the middle cerebral artery.After 2 hours of ischemia and 24 hours of reperfusion,the neurological deficits score was evaluated according to Bederson’s method.The volume of cerebral infarction in different groups was observed by 5-triphenyltetrazolium chloride(TTC)staining.And The regeneration of nerve cells in different regions of brain tissue was detected by BrdU+NeuN immunofluorescence staining in MCAO group.Culturing BMSCs from different groups and using exosome kits to extract exosomes from BMSCs and serum.Then using Transmission Electron Microscope(TEM)to detect the morphological size of extracted exosomes.The expression of Zeb2 gene in BMSCs,BMSCs’exosomes and serum exosomes were respectively examined by Reverse Transcription-Polymerase Chain Reaction(RT-PCR)and Western blot(WB)at RNA level and protein level.Results:1.After 24 hours ischemia-reperfusion,the score of neurological deficits of SD rats was significantly increased.2.We observed that after 24h ischemia-reperfusion,MCAO group’s rats had slight edema in brain tissue and pale ischemic infarct foci in one cerebral hemisphere.TTC staining results showed that there were obvious ischemic infarct foci in MCAO group and the infarct volume was significantly larger than that in Sham group.3.BrdU+NeuN immunofluorescence staining showed that In MCAO group,BrdU+/NeuN+double positive neurons were not found in SVZ,hippocampal region and cortex of rats after 24 hours ischemia-reperfusion.4.TEM showed that exosomes are round or biconcave shape small vesicles with a diameter of about 40-160 nm,which have clear edges,complete intact structure and no aggregation.5.The results of RT-PCR showed that compared with Sham group the relative expression of Zeb2 mRNA in BMSCs,BMSCs-derived exosomes and serum exosome in MCAO group showed a significant increase(all P<0.05).6.Western Blot showed that after 24 hours ischemia-reperfusion,the expression levels of Zeb2 protein in BMSCs,BMSCs-derived exosomes and serum exosomes in MCAO group increased,and the differences between the two groups were statistically significant(all P<0.05).Conclusion:1.SD rats will have obvious neurological defects,ischemic infarction foci and nerve cells damage after ischemia-reperfusion injury,.2.Zeb2 in BMSCs-derived exosomes may become a potential diagnostic and therapeutic target for ischemic stroke. |
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