The Study Of IGF-1 Gene Transfection Of Neural Stem Cells Transplantation In The Treatment Of Neonatal Rats HIBD

Neonatal hypoxic-ischemic brain damage (HIBD) is a common disease in the neonatal period, refers to a variety of reasons, mainly referring to perinatal asphyxia, caused by partial or complete cerebral hypoxia,cerebral blood flow reduction or suspension,leading to apoptosis or necrosis of a large number of nerve cells, brain edema, vesicles of periventricμlar white matter, secondary reconstruction of brain tissue, causing damage to brain structure and function, resulting in the corresponding nerve dysfunction, and severe sequelae,is the cause of acute neonatal mortality and chronic nervous system damage, one of the main reason of nervous system post neonatal disability (mental retardation,cerebral palsy and epilepsy). In recent years, with the increasing of rescue techniques, the incidence of brain injury not only did not fall, but tended to increase. Current treatment aims to relieve symptoms and control of further brain damage, poor efficacy. Its self-renewal of neural stem cells, sustained proliferation capacity and differentiation potential of the characteristics of more than replace damaged nerve cells, integrated into the host tissue of the nerve loop, inhibit neuronal apoptosis, and promote the repair of neurological function, as treatment of nerve Diseases of the ideal seed cells.Source of neural stem cells, compared to other cell sources in the treatment of nervous system cell transplantation, cord blood stem cells for its rich source,weak antigens, the low incidence rate of graft-versus-host disease,response to light, does not involve social ethics and law which has become the new source of neural stem cells. So the use of umbilical cord blood stem cells induced to differentiate into NSCs for clinical study has broad application prospects.At the same tine,neural stem cells are good carriers, using gene transfer technology, the target gene through the vector (eg, liposomes, viruses, a variety of cationic substances and calcium phosphate, etc.) was transfected into neural stem cells, their stability to be detected after expression, then the neural stem cells containing target gene were to be transplanted. Gene-modified neural stem cells can not only play a role in cell replacement and nutritional factors secreted by the target gene to improve the local environment within the brain injury to promote axonal regeneration.Insulin-like growth factor 1 (IGF-1) in the central nervous system, the whole process of development and maturation plays an important role in brain development and is necessary to promote neurotrophic and neuroprotective factor. Recent studies have found, IGF-1 for some of the central nervous system injury, neurodegenerative diseases and nerve toxicity has obvious therapeutic effect, protecting nerve cells, inhibition of apoptosis of nerve cells, reducing function after injury loss, and promoting functional reconstruction after injury.Previous studies have shown that IGF-1 factor alone or NSCs alone all can play an important role in treatment of animals HIBD model, the subject will employs NSCs transplantation and IGF-1 combination,that the IGF-1 gene transfected NSCs, by transplanting genetically engineered of the neural stem cells (NSCs-IGF-1) of the therapeutic effect of HIBD.Objective(1) The experimental study the method of cord blood derived neural stem cells isolated,cultured and differentiation, we observed the cell proliferation, differentiation.(2) To construct the expression of human IGF-1 gene plasmid vector pcDNA3.1-IGF-1, then pcDNA3.1-IGF-1 was transfected into human umbilical blood derived neural stem cells to construct genetically engineered neural stem cells of(NSCs-IGF-1).(3) The transplanting NSCs-IGF-1 into HIBD rat model, observing neural stem cells in the host brain distribution of the survival and migration, the recovery of neurological function in brain injury rats to study the situation to the clinical application of gene transfection of neural stem cell transplantation for treatment of HIBD to provide experimental basis and theoretical basis.Methods(1) By density gradient centrifugation, isolated from umbilical cord blood mononuclear cells with the cells factors of hEGF, bFGF, and B27.Induction of the differentiation of neural stem cells to observe the training process of proliferation and differentiation of cord blood mononuclear cells characteristic of immune cells detect chemical markers of neural cell-specific Nestin, NSE, GFAP expression; BrdU reagent labeled cells, BrdU-positive cells rate was determined.(2) The IGF-1 gene was extracted from the fetal liver via RT-PCR,then the product of RCR and plasmid pcDNA3.1 were purified by gel extraction. After enzyme digestion of DNA restriction enzyme-BamHⅠand HindⅢ, purified IGF-1 gene was cloned into expression plasmid vector pcDNA3.1 by T4 DNA Ligase.Recombinant plasmid was identified by DNA sequencing method and enzyme digestion of DNA restriction enzyme-BamHⅠand HindⅢ.(3) Recombinant pcDNA3.1-IGF-1 and empty plasmid were transfected into human umbilical cord blood-derived neural stem cells through lipofectin transfection. After transfection,transfected neural stem cells were filtered with neomycin (G418). The expression of IGF-1 gene in the gene transfected neural stem cells was examined by immunocytochemical method and RT-PCR.(4) 7 days 75 newborn SD rats by ligation of the left common carotid artery, and the hypoxic animal model was prepared HIBD. After modeling, the 75 rats were randomly divided into HIBD NSCs transplantation group,model control group, NSCs-IGF-1 transplant groups of 25 rats were randomly divided into further groups 1 day,7 days group,14 days group,21 days group and neurological testing group, n=5. Then 3 days before transplantation, the cells of NSCs transplantation group, and NSCs-IGF-1 transplantation group were labelled by BrdU(final concentration lμmol/ L).24 hours after modeling, adjusting the cell concentration (1×106/L), by tail vein injection of stem cell transplantation.25 without modeling, and does not transplant any cells of normal SD rats as normal control group. After modeling the line SD rats and transplanted NSCs-IGF-1 for the NSCs-IGF-1 transplant group, the SD rats after modeling the line in the same position as NSCs NSCs transplantation group, after modeling the SD rats Saline injection controls for the model control group.(5) 5 animals in each group were randomly selected, after transplantation,1 day,7 days,14 days,21 days were sacrificed, BrdU was detected by immunofluorescence the expression of the brain of transplanted cells in host survival and migration were detected. Y-maze test for detection of brain injury in the rat nervous system function and learning and memory function of the recovery.Results(1) After umbilical cord blood mononuclear cells isolated directly into hEGF, bFGF, and B27 factors induced differentiation of neural stem cell clone group obtained, and expressed neuronal specific markers Nestin, NSE and GFAP.P3 generation of neural stem cells labeled by BrdU, the positive rate of 90%.(2) IGF-1 gene was successfully extracted from the fetal liver. Recombinants pcDNA3.1-IGF-1 was proved accurate by restriction enzyme digestion and sequencing. Recombinant was transfected into human umbilical cord blood-derived neural stem cells by liposome for 24 hours,then selection cell clones appeared after 10-14 days for neomycin(G418)filtering.In umbilical cord blood-derived neural stem cells transfected by recombinant plasmid vector,the expression of IGF-1 gene was successful,which was detected by immunocytochemical method and the expression of IGF-1 mRNA was also positive while the other was negative compare with controls,which was examined by RT-PCR. (3) BrdU on neural stem cell transplantation in animal models HIBD tracing shows, BrdU-positive cells in NSCs-IGF-1 group and NSCs group were visible. Vein graft from the BrdU-positive cells can migrate to HIBD rat brain, as time goes by chemotaxis to the injury side room ependymal zone of survival.7 days after transplantation, the transplantation group compared with the NSCs, NSCs-IGF-1 transplantation BrdU-positive was significantly higher, the difference was statistically significant (P<0.05)(4) IGF-1 gene in transfected neural stem cells (NSCs-IGF-1) transplantation can improve the brain injury to better learning and memory function.Conclusions(1) Cord blood mononuclear cells can be induced into neural stem cells in vitro, umbilical cord blood may be a new source of neural stem cells.(2) IGF-1 gene can express in umbilical cord blood-derived neural stem cells transfected by recombinant plasmid vector.(3) IGF-1 gene transfected NSCs have the greater ability of proliferation.(4) NSCs-IGF-1 and simple NSCs were transplanted into HIBD neonatal rats respectively, the stem cells all can remain alive in the host brain tissue and migrate.(5) IGF-1 gene transfer of NSCs may be more apparent for brain injury recovery of neurological function in rats.(6) The genetic engineering neural stem cells with IGF-1 gene transplantation for treatment of neonatal rats HIBD is an effective treatment.