| Traumatic brain injure (TBI) is the major factor leading to invalid, especially in the young people. Discovery and isolation of neural stem cells (NSCs) has changed the conventional idea that injured nervous tissue could not be regenerated, and makes it possible to repair neural injury after TBI.The proliferation of endogenous stem cells, triggered by trauma, ischemia et al, is not sufficient enough to meet the need of neural repair. Also, direct implantation of NSCs into the wounded region could not cause an ideal therapeutic effect due to the poor local internal environment.Vascular endothelial growth factor (VEGF) promotes endothelial cell proliferation, accelerates the formation of new vessels, and increases the vasopermeability. Due to aforementioned characteristics, VEGF is widely used in ischemia of extremity and coronary arteries. Recent studies show that, besides the effect on angiogenesis, VEGF may protect neural function, promote neurogenesis, which gives the light for repairing of injured nervous tissue. However, there are still a lot of unsolved problems so far, for example, requirement of repeating injections of VEGF into the brain, very expensive cost, intracranial infectibility, insufficient expression of VEGF plasmid, and unsafely of adenovirus.We want to know if NSCs, modified with VEGF gene, have characteristics of both genes, secreting VEGF, and NSCs, replacing the wounded cells. Implantation of this modified cells into traumatic brain could facilitate the formation of collateral circulation, improve local microenvironment, get final objectives of our study to recover the function of injured brain by improving the survival of host and implanted NSCs.In view of above knowledge, this experiment would transfect in-vitro cultured NSCs by an EGFP-VEGF recombinant plasmid labled by enhanced green fluorescent protein (EGFP). We would observe expression and biological effect of VEGF in cultured NSCs, investigate its effect on NSCs proliferation and differentiation, check its function in the implanted local neural region, which determined by flow cytometry, immunohistochemistry,RT-PCR, Western blot, and laser confocal immunofluorenscence microscopy. This experiment has three sections:1. Neural stem cells isolated from embryonic rat brain were cultured, certificated, and transfected with p-EGFP-Nl plasmid.2. Recombinant plasmid p-EGFP-Nl-VEGF was constructed, certificated, transfected in the NSCs.2. Implantation of NSCs modified with VEGF into traumatic brain, and investigate its effect on traumatic injured brain. Major results and conclusions:1. Primary and secondary cultured neurosphere isolated from the embryonic rat brain had positive immuocytochemistry staining of nestin(marker of stem cells), when exposing to 10%FBS, cells began to differentiate and had positive staining of NF200(marker of neuron) and GFAP(marker of astrocyte) , which confirmed the cultured cells were NSCs with proliferation and differentiation potential.2. It was found that under microscopy some NSCs transfected with p-EGFP-Nl had strong immunofluorescent signaling. Immunofluorescent positive cells took up to 35.7 5.2% of the total transfected cells, and the immunofluorescence signal lasted for at least 3 weeks. There was no difference for the cell growth curve, between NSCs with or without transfection, indicating liposome was an ideal mediator for EGFP transfection, EGFP was a good reporter of gene transfer, and NSCs acted as a carier for the expression of exogenous gene.3. EGFP-VEGF recombinant plasmid was constructed successfully by molecular cloning methods and identified by cut, PCR and sequencing analysis.4. Transfected in-vitro cultured NSCs with plasmid p-EGFP-Nl -VEGF, immunofluorescence positive cells were found under immunofluorescence microscopy. In VEGF transfected NSCs, expression of VEGF was much higher as compared with its non-transfected control cells. Supernatant of culture media improved endothelial cells growth, and Mile's study yielded positive r...
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