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Construction and Identification of SHH-N Gene Adeno-associated Virus Vector and Its Effection on Genes Related to Proliferation in Neural Stem Cells

Author: LiuDongSheng
Tutor: ZhangBo
School: Dalian Medical University
Course: Surgery
Keywords: Neural stem cells SHH signal pathway N-myc gene Prolifratio Target gene
CLC: R329
Type: Master's thesis
Year: 2011
Downloads: 6
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Abstract

Sonic hedgehog (SHH) is a important signaling molecule in the patterning, proliferation, and cell fate determination of a broad range of cells and tissues. In the developing nervous system, SHH modulates patterning of the neural tube and regulates the differentiation and proliferation of neural stem cell . Neural stem cells(NSCs) are self-renewing, multipotent progenitor cells that reside in the subgranular zone of the dentate gyrus within the nervous system and subventricular zone of the lateral ventricle and are able to differentiate into thress major neural cell types:neuron, oligodendrocyte and astrocyte . Recently it has demonstrated that SHH is required to maintain the progenitor cell niche in the telencephalon and that exogenous SHH enhances the proliferation of Neural Stem Cells..Alough lots of mitogens,neurotrophins and other factors modulate the proliferation of NSCs,the mechanisms underlying this process are poorly understood.The precise mechanisms by which SHH promotes proliferation and tumor formation are unknown. In most cells, the transmembrane protein encoded by patched1 represses transcription of SHH target genes . When SHH binds to Patched, the repression is relieved, and a protein called Smoothened becomes activated.Smoothened activation leads, through steps that are poorly understood, to posttranslational modification and nuclear translocation of Gli-family transcription factors. Once in the nucleus, Gli proteins bind to DNA and regulate target gene transcription.Cellular competence restricts the proliferative response to SHH in vivo and provide evidence that proliferation and differentiation can be regulated separately in precursor cells of the spinal cord Adeno-associated viral vector delivery of SHH cDNA to the hippocampus elicited a 3.3-fold increase in cell proliferation. Finally, the pharmacological inhibitor of SHH signaling cyclopamine reduced hippocampal neural progenitor proliferation in vivo. The loss of hedgehog signaling also resulted in abnormalities in the dentate gyrus and olfactory bulb. Furthermore, stimulation of the hedgehog pathway in the mature brain resulted in elevated proliferation in telencephalic progenitors. These results suggest that hedgehog signaling is required to maintain progenitor cells in the postnatal telencephalon[20].In this study, our result show that(1)SHH-N gene was coincident with NCBI report.(2) pSNAV2.0 -CMV-SHH-N-IRES-EGFP expression vector and rAAV-SHH-N -EGFP vector was successful established and packaged.(3) Real time PCR was performed after SHH-N infection for 48 hour, induction of N-myc and Gli1 in rAAV- SHH-N-EGFP -treated group was enhanced compared to control group.Overexpression of N-myc promotes cell-autonomous G(1) cyclin upregulation and CGNP proliferation independent of SHH signaling[21]. Furthermore, Myc antagonism in vitro significantly decreases proliferative effects of SHH in cultured CGNPs. Together, these findings identify N-myc as a direct target of the SHH pathway that functions to regulate cell cycle progression in cerebellar granule neuron precursors.In short,We show N-myc is a direct downstream target of the SHH signal pathway in the neural stem cells. The increase in N-myc transcription stimulated by SHH suggested that N-myc might be an important mediator of SHH-induced proliferation. Although examination of sequences in and around the N-myc gene does not reveal any consensus Gli-binding sites, it is possible that such sites are present in other parts of the gene, or that SHH regulates N-myc expression through Gli-independent mechanisms.It would be interesting to investigate possible roles of N-myc in SHH signaling pathway in future studies.

Construction and Identification of SHH-N Gene Adeno-associated Virus Vector and Its Effection on Genes Related to Proliferation in Neural Stem Cells

Abstract

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