| TLX belongs to a class of orphan nuclear receptors that underlies many aspects of neural development in the CNS. TLX gene was expressing in vertebrates forebrain during embryogenesis, it can help maintaining neural progenitor cells quantities and play an important role in its differentiation into neurons,TLX gene knock-out mice its neural progenitor cells proliferative state and differentiation into neuronsability were influenced. TLX also underlies a fundamental developmental program of retinal organization and controls the generation of the proper numbers of retinal progenies and development of glial cells during the protracted period of retinogenesis. In addition, TLX gene can significantly inhibit expression of the astroglial marker glia fibrillary acidic protein (GFAP) in neural stem cells.Many stem cells such as ESCs and NSCs can be used to treat a lot of neurological disease. But the use of embryonic stem cells ESC may raise ethical issues and practical problems such as the fate of the embryonic cells in adult environment and the required immunosuppression. NSCs transplantation, indeed, is limited by the inaccessibility of its source, that is located deeply in the brain. Therefore the identification of a readily accessible source of neural cells obtainable without invasive procedures could be of a great benefit. Several non-neuronal tissues can be a source of adult stem cells capable of neuronal differentiation. Recently it has been shown that dermal multipotential stem cells is present in the dermis of mammalian skin, and if grown in vitro it preferentially forms floating spheres constituted by cells positive for fibronectin and nestin. After long term and continuous cultured, dermal multipotential stem cells maintain highly proliferative and differentiative ability, which suggests that DMSCs is a perfect"seed"cells in cell substitute therapy.Both the absence of neuronal regeneration ability and local environment factor in spinal cord injury are harmful to the repairment of injuried spinal cord tissues. The recovery of spinal cord after injury is all along a difficult problem that puzzles the neuroscientists. Numerious studies had been carried out by neuroscientists, and some encourage results had been obtained. Dermal multipotential stem cells (DMSCs) transplantation had been a prospect treatment for spinal cord injury (SCI). Deliveries of therapeutic genes are also new and promising strategies to simulate regeneration of spinal cord after injury. DMSCs engineered by gene combines the therapeutic values of DMSCs transplantation and gene delivery.In order to determin the effects of TLX gene on the proliferation and differentiation into neurons of DMSCs and see if the DMSCs and DMSCs engineered by TLX gene have the effects of promoting the regeneration and function recovery of SCI, a series of studies were carried out as follows: (1)clone rat TLX CDS sequence and built pEGFPN1 vector containing TLX gene complete CDS sequence, and examined its expression; (2)explored effects of TLX in proliferation and differentiation into neurons of DMSCs; (3) transplanted DMSCs modified by TLX gene into the area of spinal cord transverse in rats, explored effects of transplantation on promoting function recovery of spinal cord, and clarified roles of DMSCs modified by TLX gene on recovery of SCI in rats. We hope that the transplantation material- DMSCs modified by TLX gene express TLX protein eternally, and provide a possible agreeable microenvironment for regeneration and recovery of spinal cord after injury. The main results are as following:1. In this study, rat TLX full length CDS sequence was amplificatied through Reversetranscription-PCR(RT-PCR), the TLX degenerate primers were used and the total RNA in adult rat brain was the amplificative templet. Basic local alignment search tool (BLAST) search online found that the sequence was 99% similarity to the rat TLX predicted sequence. It suggested that we successfully cloned the rat TLX gene. The sequence was submitted to GeneBank , We get a Gene accession number:EU316216.2. Using RT-PCR product, the TLX cDNA was cloned to pMD18-T, then identified pMD18-T-TLX by double enzyme cutting by means of restriction enzyme BamHI and Hindâ…¢,and then the TLX fragment second-cloned to lined pEGFPN1. Then the pEGFPN1- TLX was transferred to DMSCs. The GFP expression and TLX expression was detected with fluorescence microscope and RT-PCR respectively. The results suggests that pEGFPN1-TLX was successfully constructed .In adition, the result of RT-PCR indicates that the TLX was successfully transferred to DMSCs and expressed in DMSCs.3. Using MTT we conclude that TLX can promote the proliferation of DMSCs;When DMSCs differentiated in vitro, DMSCs can be differentiated into neurons and astrocytes, and the the percent of astrocytes was higher than neuons,While DMSCs modified by TLX gene differentiated, the percent of neurons increased. TLX helped the differentiation of DMSCs to neurons, but the percent of astrocytes decreased.4. DMSCs and DMSCs modified by TLX gene were transplanted into spinal cord transverse injured site. The results indicated that DMSCs and DMSCs modified by TLX gene survived and integrated into the host spinal cord, the structure and fuction of spinal cord were recovered partially. It seems that transplantations promoted partial recovery of motor functions of spinal cord, and the effects of DMSCs modified by TLX gene group seems better than that of the DMSCs group.In summary, we successfully cloned the rat TLX gene. The sequence was submitted to GeneBank , We get a Gene accession number:EU316216. And then we built pEGFPN1 vector containing TLX gene complete sequence. Results showed TLX gene promoted the proliferation and the differentiation of DMSCs to neurons and inhibited the differentiation of DMSCs to astrocytes. The transplantation of DMSCs and DMSCs modified by TLX gene promoted recovery of structure and function of spinal cord after injury, the effects of DMSCs modified by TLX gene were a litter better than that of DMSCs. Our results indicated that TLX gene probably involved in the processs of repair of spinal cord and rebuilding of function through helped the differentiation of DMSCs to neurons and inhibited the differentiation to astrocytes. The results of this study provide some fundmental information for the clinical application of SCI..
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