Study On The Treatment Of Ischemic Stroke Model With HOM-MSCs Pretreated By Hypoxia And Ischemia

OBJECTIVE: It is proved that hOM-MSCs(Human olfactory mucosa mesenchymal stem cell)can protect mitochondrial function by miR-181 a mediated GRP78(Heat shock protein70,downstream t arget of HSP70 family)and BCL2(B cell lymphOMa-2,downstrea m target of anti apoptosis family)to protect neurons in hypoxic an d ischemic microenvironment.And verify this mechanism in animal experiments.This may provide another theoretical evidence supporti ng for MSCs treatment of ischemic stroke,and may also provide a new and feasible method for the treatment of ischemic stroke in t he future.METHODS: 1.OGD(Oxygen and glucose deprivation)neuron model;2.Extraction,culture,purification and identification of hOM-MSCs;3.Grouping: the in vitro experiment was divided into four groups:hOM-MSCs group with hypoxic ischemic pretreatment,hOM-MSCs group without hypoxic ischemic pretreatment,hOM-MSCs + miR-181 a analog group,PBS group.4.Transwell experiment.The apoptosis,ROS(reactive oxygen species)and JC-1 mitochondrial membrane potential fluorescence of neurons were detected 24 hours after co-culture;5.Make MCAO(middle crebral artery occlusion)/Reperfusion model rats;6.In vivo experimentalgroup: hOM-MSCs group with hypoxic ischemic pretreatment,hOM-MSCs group without hypoxic ischemic pretreatment,hOM-MSCs + miR-181 a analog group,model group,sham operation group,blank control group;7.The rats were randomly divided into 6 groups.The first three groups were injected with 1 × 106 cells in 0.5ml solution.Model group and sham operation group were injected with 0.5ml saline.The modified neurological deficit scale(mNSS)included exercise test?sensory experiment?loss of reflexes and abnormal movement?balance beam test.The highest score is 18,and the lowest score is 0.The lower the score,the better the function;8.TTC staining test.RESULTS:(1)MiR-181 a secretion of h OM-MSCs decreased significantly after 36 hours of ischemic and hypoxic pretreatment;miR-181 a expression of h OM-MSCs increased significantly in the simulated group;(2)BCL2 and GRP78 expression of hOM-MSCs increased significantly after 36 hours of ischemic and hypoxic pretreatment;BCL2 and GRP78 expression decreased significantly in the simulated group;(3)Apoptosis rate of cells in the ischemic and hypoxic pretreatment group(36h)was the lowest;(3)The level of oxidative stress was the lowest in ischemic and hypoxic pretreatment group(36h);(5)The fluorescence level of JC-1 was the lowest in ischemic and hypoxic pretreatment group(36h);(6)After 36 hours of ischemic and hypoxic pretreatment,hOM-MSCs could improve the neurologicalfunction of rats with ischemia-reperfusion.CONCLUSIONS :(1)hOM-MSCs can reduce the secretion of miR-181 a and increase the expression of BCL2 and GRP78 after 36 hours of ischemic and hypoxic pretreatment;(2)hOM-MSCs can reduce the oxidative stress level of hypoxic ischemic neurons,stable mitochondrial membrane potential and reduce cell apoptosis after 36 hours of ischemic and hypoxic pretreatment;(3)After 36 hours of ischemic and hypoxic pretreatment,h OM-MSCs could improve the neurological function of rats with ischemia-reperfusion.