| In long time, the injury and regeneration of central nervous system is a problem for human health, which scientists devote themselves to investigate. From the century of 19th to 20th, people found gradually that central and peripheral nerve could regenerate after injury in vertebrate and amphibian animals. But in mammalian animals, they confirmed CNS could not regenerate and peripheral nervous system could regenerate after injury. And this standpoint prevented much from therapy of nervous system disease. Although there was some progress in traditional medication and surgery therapy, but it could not get a satisfactory effect in some therapy. However, scientist never give up the research of CNS regeneration. In these years, accompanied by development of biomedicine technology and continuously deep research in CNS regeneration, it is certificated that CNS have biggish plasticity in mammalian animals. Especially in these ten years, according to the progress of stem cells' research, we can assure that CNS can regenerate in some conditions in mammalian animals including human, and newborn neurons can repair injury of CNS. Stem cells transplantation bring us a hope for therapy of CNS disease.Adenovirus served for gene expression vector in 1960's, at that time, virologist observed that adenoviral genome could hybridize with simian Vacuolating virus 40(SV40), which showed that adenoviral genome can carry heterologous gene. After this, adenovirus gradually become an important vector system, which apply to gene therapy successfully in vivo, in vitro and clinical experiment. At present, adenoviral vector that is used extensive is serum 5 adenovirus.Our research is primary culture and subculturing of neural stem cells (NSCs), and adeno-virus mediated LacZ gene transfection and expression in NSCs. We have constituted a gene therapy method that vector is NSCs. In our experiment, the structure of subependymal region and hippocampus are taken from embryo 14 to 16 days of rats. Used by serum-free cell culture technology, we have cultured NSCs successfully. Stimulated by cytokines of EGF and bFGF, NSCs can proliferate in vitro that is Nestin positive cells by immunocytochemistry. Moreover, adenovirus-mediated LacZ gene can express in NSCs. This research provide a solid foundation for NSCs-mediated gene therapy.Materials and methods1. Main reagent: HEK-293 cell strain, Adeno-XTM Expression System, DMEM, DMEM/F12 (1:1), DOSPER liposomes, X-gal, r-h EGF, r-h bFGF, B27, Nestin antibody, et al.2. Primary culture and subculturing of NSCs: the structure of subependymal region and hippocampus are taken from embryo 14 to 16 days of rats, and is digested into simple cell by 0.25% trypsin and 0.02% EDTA (1:1), then is evaluated by Nestin immunocytochemistry.3. pAdeno-LacZ virus package: By molecular cloning technique, pShuttle plasmid carrying LacZ gene is linked with Adeno-X?in vitro, which we call pAdeno-LacZ DNA. pAdeno-LacZ DNA is transfected into HEK293 cells mediated by DOSPER liposomes. When virus package is completed, pAdeno-LacZ virus infected HEK293 cells to get a great quantity of pAdeno-LacZ virus. pAdeno-LacZ virus is purified byCsCl gradient centrifugation. And then, virus titer is determinated by end-point dilution assay.4. pAdeno-LacZ virus transfection NSCs: 8 dilutions of pAdeno-LacZ virus transfect NSCs, which is stained by X-gal to observe the transfected cells. MTT technology is used to detect the survival rate of NSCs. And we can know the influence of pAdeno-LacZ virus to the growth and proliferation of NSCs.Results1. There is less NSCs in primary culture technology. We observe that many cells stick to the floor of plate at the first day, and a few of them stretch out processus. At the 5th day, the adhesive cells disrupt, and take on death phenomena, however, those non-adhesive cells suspend in culture medium, which become more and more and form some small cell colonies. Those passaged cells suspend in culture medium in regularly spherical shape and have non-prominence in... |
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