| Since Evans and Kaufman had isolated and cultured the inner cells from mouse embryos in 1981, neural stem cells have been separated, cultured, induced to differentiate and transplanted successfully. Neural stem cells are undifferentiated cells who have the ability of self-renewal and multi-potential of differentiation. They can produce neurons, astrocytes and oligodendrocytes under certain environment in vitro, which bring us a hope for therapy of injuries and regressive disorders in CNS.With the development of genetic engineering these years, people can modify the genes of neural stem cells and fulfill the purpose of its' scientific research and clinical therapy. The technology of gene transfer mediated by retrovirus was one of the most effective methods, which was established in eighties last century. After the recombination of retrovirus DNA and heterologous genes, the retroviral plasmids were transferred into package cells and recombinant defective virions were harvested. These virions transferred heterologous genes into the genome of the target cells and the genes were expressed successfully.Neural stem cells transplanted into live animal models would be alive or dead, and even integrate with host neural tissues. Effective detecting the tendency of the transplanted NSCs was the evidence of the effect of transplantation. Enhanced green fluorescent protein was found in Jellyfish, and enhanced green fluorescent protein was its' reformed type. With theunusual superiority of the EGFP, it is one of the most important cell tracers in cell biology.Our research is about primary culture and subculture of neural stem cells from embryonic rat brains in vitro, and retrovirus mediated EGFP gene marking NSCs. We also transplanted the marked NSCs into severe injured brain models of rats to observe the expression of EGFP and the function of NSCs transplantation.Materials and methods1. Main reagent: PA317 cell strain, DMEM, DMEM/F12 (1:1), B27, r-h EGF, r-h bFGF, Nestin, NSE, GFAP, EGFP antibody, pLEGFP-N1 retrovirus vector, EcoRI, XbaI, DNA Marker, Lipofectin, G418, et al.2. Primary culture and subculture of NSCs: The structure of subependymal region and hippocampus are taken from embryo 14 days of rats, and is digested into simple cell by 0.25% trypsin and 0.02% EDTA (1:1), then is incubated with serum free medium and identified with immunocytochemistry.3. Package of recombinant defective virions containing gene of EGFP: The retroviral plasmids, pLEGFP-Nl, which containing gene of EGFP were proliferated and transferred into PA317 cells with lipofectin. Resistant colonies were cloned and maintained under G418 selection, and the virus supernatants were harvested.4. Defective virions containing gene of EGFP transfected NSCs: The NSCs were cultured with the supernatants and selected under G418. Resistant NSCs colonies were cloned and detected by immunohistochemistry staining and under fluorescence microscope. MTT technology was used to detect the survival rate of infected NSCs under certain virus titre. And their differentiations were identified with immunocytochemistry too.5. NSCs marked by EGFP gene transplanted into severe injured rats brain models: Improved Feeney free-fall brain injury machine was adopted to form the severe brain injury model. A stainless needle connected with a micropump was penetrated into the injured tissues, injecting the NSCs marked by EGFP gene slowly (10ul in 5 minutes).And 15 days later, the rats' brains were removed and sectioned into coronal slices (2mm), under ice-cold circumstance. A slice contain injury field was fixed by 10% formalin, and sent to HE stain. The slices were observed under microscope and fluorescence microscope, and identified with immunocytochemistry. The marked NSCs were transplanted into subcutaneous part of nude mice, and observation was followed at 3 days, 1 week, 2 week, and 2 months later.Results1. There were less NSCs in primary culture. We observed that many cells stick to the floor of plate at the first day, and a few of them stretch out processu...
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