| Objective: To study the effects of bone marrow mesenchymal stem cells(BMSCs)-derived stem cell growth factor on the proliferation,differentiation and hypoxia tolerance of injured rat neural stem cells in vitro,and the repair of rat brain injury model in vivo.The effect provides theoretical guidance and experimental basis for the recovery of neurological function in the middle and late stages of brain trauma.Methods:(1)BMSCs were isolated and cultured,and the expression of specific surface marker proteins was determined by flow cytometry.(2)Enzyme-linked immunosorbent assay(ELISA)assay was used to detect the protein levels of BDNF,IGF-1,VEGF and NGF in BMSCs culture medium to understand the expression levels of main stem cell active factors in BMSCs conditioned medium.(3)The primary rat neural stem cells(NSCs)were isolated and cultured in environment containing trophic factors,then the growth of cell cloned spheres,and its differentiation potential to neuronal and neuroglia were observed.After establishment of oxygen sugar deprivation model,lactate dehydrogenase(LDH)content in supernatant was detected,the protective effect of nutrition factor under hypoxic stress was observed.(4)Rat model of impact brain injury was made,treated with stem cell active factors,and the general condition of rats was observed.Water maze test was used.(5)HE staining was performed for gross histological observation;dry/wet weight method was used to detect brain edema.(6)Fluorescence quantitative PCR was used to detect the expression of IL-1β,Gap43,Jun,TNF and GAP43 in brain tissue of rats;immunohistochemistry was used to detect the expression of BrdU + NeuN,BrdU + GFAP;Western Blot method was used to detect the expression of protein NF-κBp65 in brain tissue.Results:(1)the fourth generation of bone marrow-derived cells cultured in this experiment expressed the marker protein CD90 and CD105 were both higher than 95%.It was confirmed that the cultured cells were high-purity BMSCs.(2)The expression of VEGF in the 4th to 14 th generation BMSCs was maintained above 400 pg/ml,and the level of IGF-1 was maintained at 100 pg/ml-150 pg/ml.Between concentrations,NGF is maintained between 380 pg/ml-430 pg/ml,and BDNF levels are maintained between 310 pg/ml-330 pg/ml.(3)stem cells(NSCs)cultured in the culture environment containing BMSCs cell trophic factors grew faster than the NSCs culture medium alone.BMSCs cell nutrition Factors can induce NSCs to differentiate into neurons more easily,and can enhance the tolerance of NSCs to hypoxia stress.(4)the experimental animals in the treatment group also showed significant hemiplegia,and the body weight decreased significantly in the early stage of surgery.The water maze test results showed that the experimental animals in the active factor treatment group had better neurological recovery ability.(5)The results of HE staining showed that in the active factor treatment group,the cell arrangement was relatively more regular than that of the TBI group.and the brain edema level in the active factor treatment group was significantly lower than that in the TBI group.(6)The treatment of stem cell active factor could decrease the mRNA expression level of IL-1β,Jun and TNF after TBI;and decrease the mRNA expression level of the GAP43 after TBI;increase the protein expression levels of BrdU/NeuN;decrease the expression level of NF-κBp65 in brain tissue.Conclusion:(1)BMSCs cultured from passage 4 to passage 14 maintained high levels of expression of VEGF,IGF-1,NGF and BDNF,and sustained cellular trophic effects on surrounding cells.(2)BMSCs cell-active factors can promote the proliferation of NSCs cloning spheres,and induce NSCs to differentiate into neurons more easily.In addition,BMSCs cytotrophic factors can enhance the tolerance of NSCs to hypoxia stress.(3)BMSCs cell-active factors can promote the mobilization of endogenous NSCs and induce NSCs to differentiate into neurons.(4)BMSCs cell active factors can help brain trauma rats to improve learning and memory function,improve exercise capacity,reduce inflammation levels,and inhibit cell apoptosis. |
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