| BackgroundFor the past few years,stroke has become an important threat to residents’ health.In 2017,stroke was the third leading cause of death and disability among global residents.In 2010,stroke was the third leading cause of premature death in the United States,affecting nearly 800,000 people every year.According to statistics of the national health council of China in 2013,the national stroke prevalence rate was12.2‰.It can be seen that stroke is one of the main causes of death and physical disability in adult,and more than half of them are ischemic apoplexy.Although intravenous thrombolysis can effectively improve the outcome of patients with acute ischemic stroke,only a small proportion of patients can receive thrombolytic treatment due to the limitation of time window.More than two-thirds of stroke survivors have a legacy that requires complete or partial dependency on care.Therefore,it is of great significance to seek effective therapeutic methods to promote the recovery of neurological function.Pluripotent stem cells have unlimited differentiation and expansion potential,which can promote the recovery of nerve function in ischemic stroke.However,epidermal teratoma was found to be formed in ischemic brain tissue,which limits the clinical application of pluripotent stem cell therapy.Compared with pluripotent stem cells,bone marrow mononuclear cells(BMMCs)have limited ability of splitting and differentiation,but can be used safely for tissue regeneration with primary paracrine.Recent studies have shown that the histological and behavioral indicators of the animal with ischemic stroke improved after the intervention of BMMCs.A small number of clinical trials have also shown that BMMCs therapy improved patient outcomes.However,the specific mechanism of BMMCs promoting the recovery of ischemic stroke isn’t completely clear,which needs further research and discussion.Up to now,studies at home and abroad have reported that BMMCs can promote nerve function recovery by secreting neurotrophic factor and inhibiting inflammatory response.But,the changes in the number of astrocytes,neuronal growth inhibition factor and apoptosis factor after BMMCs intervention have not been reported.This study will further investigate the role and possible mechanism of BMMCs in therecovery of neurological function in rats with focal ischemic stroke.Part Ⅰ Extraction and identification of bone marrow mononuclear cells in ratsObjective To extract and isolate active and reliable bone marrow mononuclear cells(BMMCs)in rats for intervention trial.Methods The bone marrow of the femur and tibia was washed under aseptic condition,and the marrow fluid was obtained.The marrow mononuclear cells were isolated by centrifugation with lymphocyte isolation fluid.Activity of the cells was identified by0.4% trypan blue stain.The expressions of CD34,CD44,CD45 and CD90 in the isolated cells were observed by immunohistochemical staining.Results The cell viability was 93.17% after 0.4% trypan blue staining.Immunohistochemical staining shown that the mean number of CD34,CD44,CD45 and CD90 was(34.33±3.33),(27.67±2.94),(42.50±4.09)and(20.83±3.06).Conclusion The isolated bone marrow mononuclear cells have high survival rate and reliable components,which can be used for further transplantation.Part Ⅱ Effects of BMMCs on neurological function recovery and expression of GFAP,Nogo-A and Caspase-3 in rats with focal cerebral ischemiaObjective To determine the effect of bone marrow mononuclear cells(BMMCs)on the expression of glial fibrillary acidic protein(GFAP),Nogo-A and cysteinyl aspartate specific proteinase 3(Caspase-3)in adult rats with focal cerebral ischemiareperfusion,and study the mechanism of BMMCs in the process of nerve function recovery.Methods1.The right middle cerebral artery occlusion(MCAO)model of SD rats was established by using nylon thread to occlude the right middle cerebral artery for 2 h followed by a 24 h reperfusion according to zea-longa bolting method.The qualified models were selected according to the zea-longa scale.The models with scores of 1,2and 3 were selected for further study.2.Forty-eight models were divided into MCAO model group and BMMCs intervention group according to the random number table method,and each group was divided into three subgroups of 7,14 and 21 days according to the different observation time points,with 8 in each subgroup.3.After 24 h of ischemia reperfusion,BMMCs were transplanted into the rats with focal cerebral ischemia in intervention group by using stereotactic technique.4.Longa score was performed again before rats were killed at the corresponding time point.Immunohistochemistry assay were employed to examine the expression of GFAP and Nogo-A around the ischemic foci in the right frontal lobe,and cleaved caspase-3 was investigated by using Western Blot.Results1.Before the start of intervention,there was no significant difference in neurological deficit scores between the MCAO group and the BMMCs group(P=0.963>0.05).In BMMCs group,the neurological deficit scores on both 14 d and21d were less than that on 7d after ischemia reperfusion(P=<0.05).Meanwhile,the neurological deficit scores on 21 d in BMMCs group,was less than that on 14 d in BMMCs group(P<0.05),as well as less than that on 21 d after ischemia reperfusion in MCAO group(P<0.05).2.Compared with MCAO group,the rats in BMMCs group showed higher expression of GFAP at 14 and 21 days after reperfusion [(37.62± 2.45)vs(27.62±1.69)and(38.00±1.85)vs(27.25±1.83),P<0.05].3.Compared with MCAO group,the rats in BMMCs group showed lower expression of Nogo-A at 14 and 21 days after reperfusion [(28.88 ±2.64)vs(32.50±1.60)and(23.87±2.36)vs(32.00±1.85),P<0.05].Meanwhile,in BMMCs group,the expression of Nogo-A at 21 days was lower than that at 14 days after reperfusion [(23.87±2.36)vs(28.88± 2.64)].4.The expression of cleaved Caspase-3 in BMMCs group was less than that in MCAO group at 21 d after ischemia reperfusion [(0.17±0.07)vs(0.24±0.15)].Conclusion1.BMMCs treatment can improve the degree of nerve function defect in rats with focal cerebral ischemia.2.BMMCs treatment can increase the expression of GFAP,reduce the expression of Nogo-A and cleaved Caspase-3 around the ischemic foci in the right frontal lobe of rats with focal cerebral ischemia.3.It was speculated that BMMCs treatment promoted the recovery of nerve function in rats with focal cerebral ischemia,which may be related to the promotion of GFAP expression and the reduction of Nogo-A and cleaved Caspase-3 expression around the ischemic foci in the right frontal lobe. |
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