| 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 family of IGF. NOV gene is a factor that regulates development of the nervous system. Recent data indicated that NOV gene may participate in the processes of regeneration and repair after of central nervous system (CNS) injury. 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. Among them, neural stem cells (NSCs) transplantation had been a prospect treatment for spinal cord injury (SCI). NSCs can give rise to neurons, astrocytes and oligodendrocytes, then integrate into the host tissues, and repair the injury area, then promote the function recovery of spinal cord. Deliveries of therapeutic genes are also new and promising strategies to simulate regeneration of spinal cord after injury. NSCs engineered by gene combines the therapeutic values of NSCs transplantation and gene delivery. In order to determin the effect of NOV gene on the proliferation and differentiation of NSCs and see if the NSCs and NSCs engineered by NOV gene have the effects of promoting the regeneration and function recovery of SCI, a series of studies were carried out as follows: (l)built pcDNA3.1/Myc-His(+)//acZ vector containing NOV gene complete sequence, and examined its expression; (2)explored effects of NOV in proliferation and differentiation of NSCs;(3)studied effects of NOV in permeability of the gap junction(GJ) of neurons and astrocytes; (4)transplanted NSCs modified by NOV 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 NSCs modified by NOV gene on recovery of SCI in rats. We hope that the transplantation material-NSCs modified by NOV gene express NOV protein eternally, and provide a possible agreeable microenvironment for regeneration and recovery of spinal cord after injury. The main results are as following:1. The NOV gene complete sequence were synthesized by RT-PCR, and ligated into HamH I and Hind III sites of pcDNA3.1/Myc-His(+)//acZ vector. The nucleotide sequences of the cDNA were determined. The vector was led into cultured NSCs and COS-7 cells using DOTAP liposomal transfection reagent. The expression of NOV protein was detected by Western blot and immunocytochemistry.2. Conditioned medium (CM) from cultured NOV/COS-7 cells (NOV-CM) was collected, it markedly promoted the incorporation of JH-TdR in cultured NSCs, the effect was concentration-dependent, this indicated that NOV-CM promoted proliferation of NSCs. While NSCs differentiated, NOV-CM helped the differentiation of NSCs to neurons, the percent of neurons increased. While NSCs modified by NOV gene differentiated, the percent of neurons also increased.3. NOV-CM markedly promoted the incorporation of 3H-TdR in cultured astrocytes, the effect was concentration-dependent, NOV-CM stimulated GFAP expression of astrocytes, and this indicated that NOV-CM promoted proliferation and activation of astrocytes. NOV-CM caused a marked reduction of the permeability of gap junctions of neurons and astrocytes, displaying loss of dye coupling, down-regulation of expression of connexin 43(Cx 43) at the mRNA level.4. NSCs and NSCs modified by NOV gene were transplanted into spinal cord transverse injured site. The results indicated that NSCs and NSCs modified by NOV gene survived and integrated into the host spinal cord, the structure and fuction of spinal cord were recovered partially. Neurons retrogradely labeled by HRP appeared in upper segmental spinal cord. It seems that transplantations promoted partial recovery of motor functions and conduction functions of spinal cord, and the effects of NSCs modified by NOV gene group seems be...
|
PDF Full Text Request