| The recovery of central nervous system (CNS) after injury is all along a difficult problem that puzzles the neuroscientists. In the past, it was widely accepted that once CNS was injured, nothing could be done. Since recent two decades, numerous studies have been carried out by neuroscientists and it is concluded that CNS after injury can be recovered.The course of recovery based on two factors: intrinsic properties and environmental cues. So, it is aimed to promote the regeneration of neurons and provide a possible agreeable microenvironment for regeneration and recovery of CNS injury.In regenerative medicine, some encourage results have been obtained. It is well known that cell replacement therapies show particular promise in the nervous system, and transplanted embryonic stem cells (ESCs) or neural stem cells (NSCs) have been shown to promote functional recovery in animal models, for example, spinal cord injury (SCI). Many stem cells (such as ESCs and NSCs) can be used to treat a lot of neurological diseases. Although the therapeutic potential of such transplants is clear, a number of problems, such as insufficient cell number, ethical issues, immunosuppression, and so on, limit their application in clinic. In this background, a new type of stem cells called dermal multipotent stem cells (DMSCs) was discovered in adult mammalian skin. They can differentiate into cells of both neuroectodermal and mesodermal lineage, including (perhaps not limited to) neurons, glia, osteogenic cells and adipocytes. Compared with the above cells, DMSCs, which own multi-oriented differentiative potential and self-renewal capability, have an accessible, potentially autologous tissue source and can expand in vitro cell culture.So they have important therapeutic implications.Deliveries of therapeutic genes are also new and promising strategies to simulate regeneration and recovery of CNS injury. Nephroblastoma Overexpression gene ,abbreviated as NOV, is a proto-oncogene, which was discovered in 1991. NOV protein, which is encoded by NOV gene, is a kind of insulin-like growth factor (IGF) binding protein (IGFBP), and one of the superfamily of IGF. NOV gene is a factor that regulates development of the nervous system. Recent data indicated that NOV gene can provide a possible agreeable microenvironment in regenerative process by IGF-depedent or IGF-indepedent ways. NOV gene may participate in the processes of regeneration and repair after of CNS injury.DMSCs engineered by NOV gene combines with the therapeutic values of DMSCs transplantation and gene delivery. In order to identify the properties of DMSCs and determinate the effect of NOV gene on the proliferation and differentiation of DMSCs, a series of studies were carried out as follows: (1) Construct pEGFP-N1 vector containing NOV gene complete sequence, and examine its expression; (2) Isolate and identify DMSCs from neonatal rat; (3) Study NOV effects on proliferation and differentiation of DMSCs. We hope that the transplantation material—DMSCs modified by NOV gene may express NOV protein eternally, and provide a possible agreeable microenvironment for regeneration and recovery of CNS injury.The main results and conclusions are summarized as follows:1. The NOV gene complete sequence was amplified from the total RNA of normal rat brain tissue by RT-PCR and cloned into pMD-19T vector (NOV-T), then the NOV fragment ligated into HamH I and Hind III sites of eukaryotic expression vector pEGFP-N1 (NOV-N1). The cloned insert in both NOV-T and NOV-N1 were identified by double digestion of the recombinant plasmid with restriction enzymes Hind III and BamH I and by nucleotide sequences. The results suggested that eukaryotic expression vector containing coding region of NOV gene was successfully constructed. The eukaryotic expression vector containing coding region of NOV gene can provide a strong molecular tool for the study of the effect of NOV gene.2. Dermis of neonatal rat were digested with 0.25% trypsin and cultured for 6h in vitro, then the adherent cells were harvested and subcultured. After the fourth subculture, the cells were used for experiments. Cell cycle was analyzed by measuring DNA content with FAC-Scan flow cytometer and cell proliferation activity was tested by MTT method. Differentiated cells were identified by immunocytochemical ABC technique and specific staining. More than 86% of DMSCs were in the G0/G1 phases and these cells display multi-lineage differentiation potential, producing both neural and mesodermal progeny. The results showed Mesenchymal stem cells with multi-lineage differentiation potential exist in neonatal rat dermis and dermis may be another important source of mesenchymal stem cells.3. The recombinant plasmid of NOV gene was transfected into DMSCs at first time with liposome. The expression of NOV gene was observed under Fluorescent microscope and detected by RT-PCR. The ratio of transfected cells was about 30%. DMSCs modified by NOV gene (NOV-DMSCs) offer a chance to study the function of NOV in vivo and DMSCs could be a novel carrier of the NOV gene.4. In order to compare the proliferative capability with NOV-DMSCs and DMSCs, cell cycle was detected by FCM and cell proliferation activity was tested by MTT method.The results indicated NOV-DMSCs in G0/G1 phase decreased while in G2/M and S phase increased than DMSCs did. Meanwhile NOV- DMSCs had shorter doubing time and faster growth velocity. Statistical analysis demonstrated that NOV-DMSCs had higher proliferative capability than DMSCs did.5. Conditioned medium (CM) from cultured NOV-DMSCs (NOV-CM) was collected, and it markedly promoted proliferation of DMSCs. When DMSCs differentiated, NOV-CM helped the differentiation of DMSCs to neurons, and the percent of neurons increased. While DMSCs modified by NOV gene differentiated, the percent of neurons also increased. This result suggested that both NOV gene and protein can improve the neural-oriented differentiation of dermal multipotent stem cells in rats.In summary, dermis mesenchymal stem cells (DMSCs) were isolated from the neonatal rat skin, cultured and identified by their multilineage differentiation capacity. Eukaryotic expression vector containing coding region of NOV gene was successfully constructed and transfected into DMSCs at first time with liposome. Further sdudies showed that both NOV gene and protein could promote the proliferative capability of DMSCs and help their neural-oriented differentiation. The results suggested that not only DMSCs could be a novel carrier of the NOV gene but also DMSCs engineered by NOV gene could combine the therapeutic values of cell transplantation and gene delivery. The results of this study provided some fundmental information for the clinical application of regeneration and recovery of CNS injury.
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