Research On The Function Of HUC-MSCs-pIGF-1 In Neural Repairation Of Brain Injury

Brain injury is caused by external factors in the head of a serious trauma and mortality rate is between 4%-7%.The mortality rate of severe brain injury is more than 50%,and the prognosis is often poor.Common brain injury includes neonatal hypoxic ischemic brain damage(neonatal hypoxic ischemic brain damage,NHIBD),hypoxia ischemia brain damage(plateau hypoxic ischemic brain damage,PHIBD),radiation brain injury(radiation brain injury,RBI)and so on.Neonatal hypoxic ischemic brain injury is the main cause of chronic nervous system injury and neonatal acute death.The disease case fatality rate is high and the disability rate is high.With improvement of the Iife support technology and neonatal rescue level,severe asphyxia and premature infants with brain injury infants survival rate is greatly improved,the neonatal hypoxic ischemic encephalopathy(HIE)prevalence did not reduce,however increased.This situation has brought serious impact on children’s physical and mental development and quality of life.Plateau hypoxia ischemia brain damage occurs in the high altitude plateau idiopathic disease,due to the acute onset,more complications,severe illness,treatment is always not timely and rapid onset of mortality rate is very high.Therefore,it is a serious threat to the high altitude population lives and safety of the plateau.Radiation brain injury refers to the side effects of radiation therapy in the treatment of brain disease or human contact high dose radiation produced which caused by acute and chronic toxicity.The disease often make the patient produces a lot of complications such as fatigue,headache,vomiting,diarrhea and other reaction,and accompanied by immune function degradation,the decline in the number of white blood cells and so on,which resulted some patients had to give up continue to accept therapy.The mechanism of radiation-induced brain injury is not very clear,so it lacks of effective treatment and drug treatment.Neurons and glial cells in the mammalian brain was confirmed to have the ability of self-renewal 20 years ago,and now the research on neurobiology become hotspot in the research of central nervous system injury,especially cell replacement therapy has become a focal point of commercial activities and research institutions.More and more evidence suggests that stem cell transplantation may be one of the most promising treatment strategies for the functional impairment after brain injury.Cell replacement therapy originates from following treatment concept,the loss of nerve function are related to disease or trauma can be through the introduction of new cells to replace the loss of neurons and glial cells,or introduced the cells can be through the neurotrophic effect to increase the damage of God by cell plasticity and functional recovery and survival rate to play a role.Up to now,various types of stem cells,including neural stem cells,embryonic stem cells and mesenchymal stem cells have been used to study the treatment of central nervous system injury.The application of Embryonic stem cells(ESCs)exists many problems such as lack of obvious moral disorders,fetal tissue source and tumor formation after transplantation although it act a role on rat brain injury function recovery,which limits its application in the human body.Recent studies have shown that pluripotent stem cells iPS,which induces pluripotent stem cells,but it also has the risk of tumor formation in the body.Neural stem cells(NSC)is a kind of used to transplantation in the treatment of cerebral ideal seed cell damage,it can secrete various neurotrophic factors and can differentiate into neural cells,but a large number of nerve stem cells as a source of problems still limits its application.Human mesenchymal stem cells(MSC)are considered to be a good candidate for the treatment of brain injury.Bone marrow mesenchymal stem cells(BM-MSC)are widely used in clinical research and experiment.But the bone marrow is a highly invasive process,there is the risk of infection,and its ability to amplify and differentiation of the potential will be decreased with the increase of age.Therefore,it has become a hot research topic to find alternative BM-MSC cells.The discovery of human umbilical cord mesenchymal stem cells(UC-MSCs)has brought new hope to patients with brain injury.Umbilical cord mesenchymal stem cells have the potential of multi differentiation.In certain condition it can be induced differentiated into adipose cells,myocardial cells,bone cells,cartilage cells and nerve cells,which is the ideal tissue engineering seed cells?with a strong differentiation and proliferation ability.And umbilical cord mesenchymal stem cell has the following advantages:they are more primitive,have stronger ability of proliferation and differentiation;acquisition and minimal invasive;weak immunogenicity;can be easily isolated;safety.At present*umbilical cord mesenchymal stem cells have become a hot research at home and abroad.For the application of MSCs research mainly concentrated in the following two aspects:one is by culture in vitro and the great expansion of MSCs and directly transplanted into the injury site of nervous system;the second is the MSCs as delivery vectors,will have therapeutic effect of specific gene was cloned into MSCs and the production of genetically engineered neural stem cells?by transplanting to the dual effect of gene therapy and cell replacement.Insulin-like growth factor-1(IGF-1)1s a kind of polypeptide hormone,which is composed of 70 amino acids,and the molecular weight is 7649.It comes from many cells in vivo,through autocrine,beside the secretion and endocrine mechanism and combined with IGF-1 receptor(IGF I R)to play a variety of physiological functions.In recent years,the study found that IGF-1 can play an important role on the brain development,growth and myelin formation,or potential with the mitogen to in vitro induced blood vessels and nerve cells of endothelial cell differentiation,proliferation and regulation of cell activity,many central nervous system diseases plays a protective effect,so more and more attention from domestic and foreign scholars focus the attention on IGF-1.Gene modified MSCs treatment is a way of gene therapy which according to gene modified neural stem cells expressing exogenous gene,then transplanted into the damaged parts,which produces a large number of neurotrophie factor treatment to prevent neuronal death and promote nerve regeneration and functional recovery of a treatment method.In this study,we choose IGF-1 to carry on the genetic modification of MSCs gene engineering umbilical cord mesenchymal stem cells,and the role and mechanism of brain injury repair by cell transplantation is still not reported.The aim of this study is to seek new sources of MSCs,the structural gene modified umbilical cord mesenchymal stem cell hUC-MSCs-pIGF-1,through the transplantation hUC-MSCs-pIGF-11 NHIBD,NHIBD,RBI rat model and RBI Tibet mini pig model was conducted to evaluate the effect and to investigate the gene modified umbilical cord mesenchymal stem cell hUC-MSCs-pIGF-1 effects on brain injury repair,and treatment of brain injury in clinical application hUC-MSCs-pIGF-1 lay the theoretical foundation.Objective1 Study the function of IGF-1 gene of Tibet minipigs in order to provide molecular basis and experimental basis for the next step.2 Study of human umbilical cord derived mesenchymal stem cells cultured in vitro and identified and observe of umbilical cord mesenchymal stem cells differentiation and proliferation in vitro culture system.3 Chemical synthesis method to construct plasmid pIRES2-EGFP-IGF-1 and according to slow virus transfection to establish the hUC-MSCs-pIGF-1 stem cell line after gene modification,which lay the foundation for the treatment of brain injury.4 Establish SD rat model of neonatal hypoxia ischemia model,high altitude hypoxia ischemia model,radiation brain injury model and Tibet minipigs radiation brain injury animal model.5 Investigate the function of hUC-MSCs-pIGF-1 in brain injury and provide theoretical support for clinical application of gene modified stem cells transplantation for the treatment of brain.Method1 PCR-SSCP method and PCR sequencing were ued to find the type and site of SNP of the IGF-1 gene in Tibet minipig.At the same time,the IGF-1 gene of Tibet mini pigs were cloned and sequenced,and the IGF-1 gene of different ages in Tibet mini pigs in the brain relative expression was determined by real-time fluorescence quantitative PCR.2 Human umbilical cord mesenchymal stem cell isolation and identification:MSCs purchase from Guangdong Province cord blood hematopoietic stem cell bank,1 x 106/ml density inoculated into culture flasks,containing B27,EGF and bFGF serum free DMEM/F12 and 37 ℃ under the condition of cell culture,half the amount of 3 days was changed.Cells were cultured up to 7 days,mesenchymal stem cells collected by centrifugation,combined with the use of trypsin digestion and mechanical method were passaged to 5 × 105/ml then stem cells were seeded on new culture bottle,37℃ and 5%to culture.MSCs containing 20%fetal bovine serum DMEM/F12 culture medium.Inverted microscope to observe cell growth dynamics.Flow cytometry was used to detect the umbilical cord mesenchymal stem cells CD90 FITC,CD 105 PE,CD73 PE,CD45 FITC,CD34 surface antigen detection.3 Plasmid pIRES2-EGFP-IGF-1 was synthesized by gene synthesis and sequenced.Human umbilical cord mesenchymal stem cells were transfected by slow virus,gene modified mesenchymal stem cells hUC-MSCs-pIGF-1 were established,and the expression of IGF-1 was detected by RT-PCR in vitro.4 By ligation of the unilateral carotid artery to construct the model of neonatal SD rats with hypoxic ischemic encephalopathy,the model was successfully constructed and injected into the left side of the brain with MSCs.85 7d SPF SD rats,weight 18-22gwere chosed.Among them,20were normal control group,65 were made of HIBD animal model.After operation,the survival of 60 rats were randomly divided into three groups:model control group,MSCs transplantation group and MSCs-IGF-1 transplantation group,each group20 each group to random is divided into the following five groups:5d group,7d group,14d group,21d group and nerve motor function testing group,3 rats in each group.The behavior(water maze)detection method for all animals were detected;hematoxylin eosin staining was used to observe the ischemic cell morphology and structure;nestin immunocytochemical staining was used to observe the mesenchymal stem cell proliferation,differentiation,and in the rat brain distribution;BrdU fluorescence labeling method to observe the expression of positive cells in the lateral ventricle;statistical region positive cells.5 48 10d SD rats were randomly divided into three groups:control group(n=8),2000 m altitude group(n=8)and 4000 m altitude group(n=8),6000m altitude group(n=24),then 6000m group was divided into experimental group,MSCs transplantation group and MSCs+IGF-1 transplantation group,each group 8 only.Low-pressure experimental group rats were placed in plateau environment simulation chamber production plateau neonate hypoxic ischemic brain damage(HIBD model)with the method of movement,movement for the cabin swimming bath for 60 minutes per day of swimming and in hypobaric environment simulation cabin life time a day not less than 20 hours.Each group were scored by Zea Longa scoring standard in 3 7,11 and 15d.Scanning electron microscope observation of the morphology of red blood cells.The brain tissues were taken for HE staining and TTC staining in each group.TTC observed changes in the area of infarct;nestin immunocytochemical staining was used to observe the mesenchymal stem cell proliferation,differentiation,and distribution in the rat brain;BrdU fluorescence microscopy was used to observe the expression of positive cells in the lateral ventricle;statistical region positive cells.6 54 adult male SD rats were randomly divided into 4 experimental groups and control group.In each group,9 rats received 2Gy,SGy and 10Gy after 3%intraperitoneal injection.The 1OGy group was divided into experimental group,MSCs transplantation group and MSCs+IGF-1 transplantation groupP 9 rats in each group.And they were sacrificed at 72h,then observed brain tissue pathology.Red blood cell morphology observed under scanning electron microscope.Nestin immunocytochemical staining was used to observe the mesenchymal stem cell proliferation,differentiation,and brain distribution;BrdU fluorescence microscopy was used to observe the expression of positive cells in the lateral ventricle;statistical region positive cells.7 36 adult male Tibet minipigs were randomly divided into the experimental group and the control group.The control group 9,the experimental group 27wwas received 10 Gy single X-ray irradiation.The experimental group of Tibet minipigs were divided into 3 subgroups,each group 9,respectively,after the radiation of 6h,24h,PET/CT was scaned after 72h and then was sacrificed for tissue pathology and ultrastructural observation of brain tissue.The expression of IGF-1 and IGF-1R in brain were detected by immunohistochemistry.Results1 There is 1 mutation sites in IGF-1 of Tibet miniature pig T40C.Comparing Tibet mini pigs IGF-1 gene coding region with the GenBank search to porcine IGF-1 Gene cDNA sequence of alignment analysis showed that,had G→A、C→T mutation at 440,455pb.The site caused by mutations in the corresponding amino acid changes,respectively by histidine into the arginine,leucine into serine.2 hUC-MSC after the first passage of the cell growth rate is faster than the primary cells,the first 3d it can reach 80%confluence,then continue to subculture.When the cells reached the third generation,the cell morphology was basically the same.Cells after cryopreservation resuscitation rate reached 90%,after the recovery of cells had no obvious change.Detection of cell surface antigen hUC-MSCs expression of mesenchymal stem cell surface antigens(CD90,CD105,CD73),the expression of surface antigen in blood and endothelial system(CD34 and CD45).That passage MSC with mesenchymal stem cell antigen characteristics.3 The destination of IGF-1 gene and plasmid pIRES2-EGFP connected product pIRES2-EGFP-IGF-1 after XhoI/EcoRI double enzyme digestion,1%agarose gel electrophoresis display in 5236bP and 579bP at each have a bright band results with a plasmid vector and target gene size consistent.It is proved that the constructed vector pIRES2-EGFP is right size and in the right direction.At the same time,the recombinant plasmid was sequenced to find that the target gene was in accordance with the genebank sequence and the expression vector pIRES2-EGFP-IGF-1 was successfully constructed.pIRES2-EGFP-IGF-1 plasmid was transfected into the umbilical cord blood mesenchymal stem cells for 48 hours,and the fluorescence confocal microscopy showed that the cells were obviously transfected with GFP.Real time fluorescent quantitative PCR showed that the relative expression of IGF-1 after transfection was significantly different(P<0.05)>the expression was up-regulated,and the expression of umbilical cord blood mesenchymal stem cells(MSCs)was higher in pIRES2-EGFP-IGF-1 transfected cells.4 The results of HE staining and water maze showed that the HIBD model could be successfully constructed by unilateral ligation of common carotid arteries and low oxygen treatment.After hUC-MSC-pIGF-1 transplantation animal model of HIBD,by immunofluorescence on BrdU tracing results show that BrdU positive cells from MSCs transplantation group and MSCs-IGF-1 transplantation group can be observed at 7 d after transplantation,MSCs transplantation between group,or MSCs-IGF-1 in the transplantation group were compared,BrdU positive rate difference significantly.The Nestin immunohistochemistry method suggested that the Nestin positive cells in MSCs-IGF-1 transplantation group and MSCs transplantation group increased rapidly,and reached the peak at 14d after transplantation,and then decreased gradually.After 7d transplantation,the expression of Nestin positive cells in the four groups were significantly different between the two two groups.The results of water maze showed that MSCs-IGF-1 transplantation group was significantly better than the model control group.5 HE staining,neurological score,TTC staining and blood cell scanning electron microscope results show that plateau combined with exercise can be successfully constructed plateau hypoxia ischemia brain injury animal model,and most notably in 6000m altitude of damage in the brain.hUC-MSC-pIGF-1 animal models of brain injury after transplantation of the plateau can find BrdU positive cells in the elevation of 6000m group MSCs transplantation group and MSCs-IGF-1 transplantation group.After 11d transplantation,the BrdU positive rate was significantly different between the MSCs group and the MSCs-IGF-1 group.After 7d transplantation,the expression of Nestin positive cells in the four groups were significantly different between the two groups.6 HE staining and blood cell scanning electron microscope results show that when the radiation dose is 5Gy,rats can appear obvious clinical symptoms,but brain HE staining results suggest that part of the brain injury is lighter.But when radiation dose is 10 Gy dose,the typical pathological can be found.Therefore,10Gy was chosen as the model of radiation brain injury criteria.BrdU positive cells from MSCs transplantation group and MSCs-IGF-1 transplantation group can be observed 48 hours after transplantation,MSCs transplantation between group and MSCs-IGF-1 in the transplantation group were compared,there is significant differences in BrdU positive rate and the four groups of nestin positive cells expression had significant difference.7 The 10 Gy was chosed to establish whole-body radiation model,HE staining and scanning electron microscopy results were visible radiation after 72h lymph node was destroyed,PET/CT results show that in 10 Gy radiation dose on brain FDG uptake showed significant differences,the SUV values and is proportional to the time.Immunohistochemistry results showed that after radiation injury after lOGy irradiation,compared with the control group,Tibet minipig brain tissue IGF-1 expression level significantly reduced(P<0.05),and IGF-1 R expression levels compared with the control group no significant difference(P>0.05),the results showed that IGF-1 in Tibet minipigs brain injury animal model is an important role to carry out cell transplantation in minipigs for the next step.