Effects Of Over-expression Of HIF-1Alpha In Bone Marrow-derived Mesenchymal Stem Cells On Traumatic Brain Injury

Background:Traumatic brain injury(TBI)is one of the most common diseases in neurosurgery.It is usually caused by mechanical damage to the brain directly or indirectly,with a high rates of disability and fatality.Statistics in recent years show that with the acceleration of urbanization and industrialization,the incidence of brain injury is also increasing year by year.Although the improvement of modern medical technology and medical level has made breakthroughs in the diagnosis and treatment of clinical brain injury and related basic research,the mortality and disability rates of patients caused by this disease remain high.Therefore,it is extremely important to seek effective TBI treatment.Bone marrow mesenchymal stem cells(BM-MSCs)as a type of Mesenchymal stem cells(MSCs)are pluripotent stem cells that have strong self-renewal ability and differentiation potential.BM-MSCs are ideal seed cells for the treatment of central nervous system injury,and the mechanism of action is related to the strong differentiation of BM-MSCs.BM-MSCs can differentiate into neurons in host neural tissue,and the differentiated neurons have corresponding biological activities,which can replace dead neurons and promote the recovery of central nervous system function.After transplantation of BM-MSCs into the host,they can survive and have the ability to differentiate into neural cells,and BM-MSCs can also migrate to the central nervous system injury area and participate the repair process.BM-MSCs transplantation has been shown to play a beneficial role in central nervous system injury,including traumatic brain injury.After transplantation,BM-MSCs can promote neurological recovery,increase endogenous cell proliferation,promote angiogenesis,reduce lesions,etc.In addition,in order to enhance the therapeutic effect of cell transplantation,BM-MSCs can be engineered to over-express genes that are beneficial to the treatment.The treatment of BM-MSCs has been one of the focuses of TBI cell therapy.How to make BM-MSCs over-expression genes that are beneficial to the treatment is a new direction of TBI treatment.Using BM-MSCs as an effective gene carrier,BM-MSCs are transfected with a virus to enable BM-MSCs to over-express beneficial therapeutic genes.The transfected BM-MSCs are then implanted into the brain to promote the therapeutic effect of cell transplantation.Some scholars at home and abroad have applied gene therapy and stem cell transplantation to TBI model animals and achieved some gratifying results.However,there is no relevant study on the treatment of TBI by modifying the over-expression of BM-MSCs HIF-1? gene.Studies have shown that hypoxia inducible factor-la(HIF-1?)plays an important role in cellular responses caused by hypoxia.HIF-1? is a transcription factor that regulates the transcription process of VEGF and EPO,which promotes angiogenesis,effectively regulates the glycolytic pathway,promotes erythropoiesis,enhances cell viability,and ultimately exerts neuroprotective effects.VEGF is a mitogenic source of endothelial cells that can recognize and bind to tyrosine kinase receptors,and then playing a role in promoting endothelial cell proliferation,migration and new vascular formation.It has been reported that VEGF can also stimulate the proliferation of neurosomatic cells.Other studies have found that the use of exogenous VEGF on traumatic brain injury mouse models can significantly increase the number of proliferating cells in the subventricular region and surrounding cortex,while also reducing the volume of lesions after traumatic brain damage.EPO is a hematopoietic growth factor in the biological body that can increase the use of oxygen during hypoxia in tissue cells.Other studies have confirmed that EPO plays an important role in angiogenesis and neurogenesis.Past studies have found that EPO treatment can effectively reduce damage to hippocampus neurons and increase angiogenesis and neurogenesis in damaged cortex and hippocampus in rats with traumatic brain injury.Current research has confirmed that mesenchymal stem cell transplantation has a positive effect on the treatment and function of central nervous system injury.There is a close relationship between HIF-1? level and hypoxia.A large number of studies have confirmed that HIF-1? mRNA and protein levels are significantly up-regulated after traumatic brain injury,suggesting that HIF-1? has protective effects on nerves.However,there are synergies between MSCs and HIF-1? that need to be further verified.In this experimen,we first selected BM-MSCs,non-other sources of MSCs,as stem cells for treatment and implanted in traumatic brain injury mouse models.The purpose of this study was to investigate whether BM-MSCs could repair the lesions of TBI model mice and whether BM-MSCs that over-expressed HIF-la gene could promote the above effects.Objectives:The treatment of BM-MSCs has been one of the focuses of TBI cell therapy.How to make BM-MSCs over-expression genes that are beneficial to the treatment is a new direction of TBI treatment.This experiment investigated whether BM-MSCs could repair the lesions of TBI model mice and whether BM-MSCs that over-expressed HIF-la gene could promote the above effects.1.To explore the possibility of over-expression of HIF-1? gene in BM-MSCs by adenovirus transfection;2.To investigate whether BM-MSCs can be used as planting cells in TBI model mice;3.To investigate the behavioral changes,neurogenesis and cell proliferation of TBI model mice after BM-MSCs were transplanted into TBI model mice;4.To investigate the changes in brain tissue volume.brain water content and expression of mRNA and protein in VEGF and EPO at various stages after BM-MSCs were transplanted into TBI model mice;5.To investigate the behavioral changes.neurogenesis.and cell proliferation after the BM-MSCs of over-expression HIF-1? were transplanted into TBI model mice;6.To investigate the changes in brain volume,brain water content,and the expression of mRNA and proteins in VEGF and EPO at various times after the BM-MSCs of over-expression HIF-1? were transplanted into TBI model mice Animals and methods:The adenoviral vector containing HIF-1? and the corresponding control vector were designed and constructed,and they were transfected into 293 cells respectively.To confirm the successful construction of BM-MSCs of over-expression HIF-1?,the adenovirus in the cell culture supernatant was collected and infected with BM-MSCs to detect the expression of HIF-? mRNA and protein in the cells.We used several BALB/c mice(6-8 weeks old)of similar weight as experimental animals.And the mouse model of traumatic brain injury was established by brain cortical impact method.After the mouse is fully awake,the neurologic function of the mice was evaluated by mNSS.The score of 7-12 is divided into moderate brain damage and indicates successful modelingAfter 6 hours of model establishment,HIF-1? overexpressed BM-MSCs were injected into the tail vein.At the same time,a blank control group,a saline-injected control group,and BM-MSCs injected group were established.In the group,the mice were tested for modified neurological deficit score(mNSS)at 1,3,7,and 14 days.Mice were sacrificed on the 7th day after TBI establishment to detect brain water content and lesion volume.Immunofluorescence staining of BrdU and BrdU/NeuN was used to detect the number of BrdU+and BrdU/NeuN+cells in mouse brain tissue,and the effect of HIF-la overexpressing BM-MSCs on cell proliferation and neurogenesis after TBI was determined.The expression of VEGF,EPO mRNA and protein in brain tissue of each group of mice was detected.All statistical analysis in this study was carried out using SPSS22.0 software.The data results are expressed as(mean±standard deviation)(x±s).Chi-square test was used to compare the two groups,and one-way ANOVA of Tukey test was used to compare the parameters of multiple groups.P<0.05 is considered to have significant statistical difference.Results:1.Hyperexpression of HIF-1? in BM-MSCsAfter we transfected HIF-1?nto BM-MSCs.the expression of HIF-1? mRNA and protein in each group was detected by RT-PCR and protein immunoimprinting tests respectively.The results are shown in Figures 1 and 2.Figure 1 shows that the HIF-lamRNA of the Ad.HIF-1? group was significantly higher than that of the.control group and the Ad.Null group.Figure 2 shows that the expression of HIF-1?protein in the Ad.HIF-1? group is the highest,and the quantitative analysis of proteins further shows that the content of HIF-1? protein in the Ad.HIF-1? group is about 3.5 times that of the control group and the Ad.Null group.These results confirmed that HIF-1? was successfully transferred to BM-MSCs and led to an increase in HIF-la protein levels.That is,the BM-MSCs that HIF-1? is over-expressed have been successfully constructed.2.Changes of nerve function and brain injury after TBI mouse transplantationTBI model mice were injected into BM-MSCs that expressed HIF-1?.and their neurological changes were evaluated by mNSS score.The mNSS score of TBI model mice was evaluated after 1,3,7,10,14days of transplantation.In the four groups(blank control group,Vehicle,BM-MSC group and HIF-1? BM-MSC group),We found that the mNSS score of the TBI model mice expressing HIF-1? in the HIF-1? BM-MSC group was the most significantly improved,suggesting that the nerve function was most restored,followed by the BM-MSC group(as shown in Figure 3).In the end,the mNss scores of the HIF-1? BM-MSC group and the BM-MSC group also showed significant differences.These results show that BM-MSCs can improve the nervefunction of TBI model mice,and over-expressed HIF-la can promote this effect.Each group of mice was taken brain after death to measure brain injury volume and brain water content.In the four groups.the lesion volume and water content of the Vehicle group were the largest,the BM-MSC group and the HIF-1? BM-MSC group decreased in turn,and the brain tissue injury volume was synchronized with the water content of the damaged brain tissue(Fig.4.5).The experimental results indicate that BM-MSCs transplantation can reduce brain injury volume and reduce brain edema,and the over-expression of HIF-1? in BM-MSCs can enhance the protective effect of BM-MSCs on TBI induced brain injury.3.Changes of neurogenesis and cell value added after TBI mouse transplantationTBI mainly causes brain cell loss and nerve necrosis.After 7 days of transplantation in TBI mice,the nerves were stained to assess the effect of transplanted BM-MSCs on the value added of neurons and nerve cells.In order to analyze angiogenesis,BrdU+cells are counted in the damage boundary area and dentate gyrus.In order to analyze neurogenesis,we calculated the BrdU+cells and NeuN/BrdU+colabeled cells in the dentate Gyrus and its subregions.Quantification of positive cells showed that the BM-MSC group had more positive cells,almost twice as many as the Vehicle group(as shown in Figure 6).However,there were significant differences in the number of positive cells in the HIF-1? BM-MSC group compared to the BM-MSC group(as shown in Figure 7).These results suggest that BM-MSCs transplantation can cause an increase in the number of positive cells associated with the growth of neurons in the damaged area,and this effect is most obvious in the HIF-1?BM-MSC group.It means that the expression of HIF-1? can promote the regeneration of neurons and the increase of nerve cells in TBI model mice.4.Changes of expression levels of VEGF,EPO mRNA and protein in TBI mice after transplantationSome experiments have pointed out that BM-MSCs play a role in promoting nerve function recovery by promoting angiogenesis and improving microcirculation in damaged areas.To verify whether BM-MSCs have the effect of promoting angiogenesis,We tested the expression levels of mRNA and protein in VEGF and EPO by RT-PCR and protein immunoassay.The results showed that the expression levels of VEGF.EPO mRNA and protein in BM-MSC group and HIF-1?BM-MSC group were significantly higher than those in Vehicle group(P<0.05),and the difference between the HIF-1? BM-MSC group is more obvious(as shown in Figures 8 and 9).Therefore,the hyperexpression of HIF-1? an enhance the induction function of BM-MSC on VEGF and EPO expression.Conclusion:1.After BM-MSCs were transplanted into the brain of TBI mice,the mNSS score decreased,cell value added and neurogenesis increased,suggesting that it could be used as a planting cell in TBI model mice and could repair nerve effects;2.After BM-MSCs were implanted into the brain of TBI mice,the lesion volume and brain water content of TBI mice decreased,and the expression levels of mRNA and protein of VEGF,EPO in the same side cortex increased.It is suggested that BM-MSCs can reduce brain damage by stimulating angiogenesis and neurogenesis after transplantation.3.After the BM-MSCs of over-expres HIF-1? was transplanted into the brain of TBI model mice,the mNSS score was further reduced,and cell proliferation and neurogenesis continued to increase.At the same time,the decrease in the volume of lesions,brain moisture in mouse brain tissue and the increase in the expression levels of mRNA,and proteins of VEGF,EPO were significantly higher than those of BM-MSCs,All that demonstrate that BM-MSCs modified by HIF-1? gene had higher proliferative activity and could significantly improve the treatment effect of BM-MSCs.4.The over-expression of HIF-1? in BM-MSCs transplantation can be used as a new method for the treatment of traumatic brain injury.lt can also be used as a scientific research basis for the combined treatment of cells and genes of ischemic hypoxic encephalopathy.At the same time.it is suggested that we can increase the beneficial genes in MSCs through virus infection and other legal persons to achieve the purpose of improving the therapeutic effect.