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Construction of cDNA Subtractive Library for Lin~-CD34~- Cells and Research on Antiapoptotic Effect of Humanin in K562 Cells

Author: WangDongMei
Tutor: PeiXueTao
School: PLA Military Academy of Medical Sciences
Course: Pathophysiology
Keywords: Lin-CD34- cells Suppression subtractive hybridization Humanin K562 cells Mitogen-activated protein kinase
CLC: R346
Type: PhD thesis
Year: 2003
Downloads: 75
Quote: 0
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Abstract


CD 34 is a transmembrane salivary mucin molecule, 1% -3% of the human bone marrow cell surface expression. Because the bone marrow in CD of 34 cells bear most of the hematopoietic vitality CD 34 molecules has been considered a positive sign of hematopoietic stem cells . Basic research and clinical applications are based on the CD 34 cell enrichment screening of hematopoietic stem cells. However, in recent years, the application of research in fetal sheep and immunodeficient mice transplantation model showed that the group does not express faculties nor specific markers and to express CD 34 molecules Lin - CD the 34 - cell transplantation conditions recipients can rebuild long-term multi-lineage hematopoiesis and immune function. Further studies showed that they have many different CD 34 hematopoietic stem / progenitor cell characteristics: First, Lin - CD 34 - cell transplant recipients, more ability to rebuild long-term hematopoietic and immune function reconstruction than the CD 34 cell transplant more quickly a higher proportion of the formation of chimeras; Second the in vitro, the CD , 34 - cells with highly proliferative and differentiation to produce a large number of CD 34 cell potential, which can make up for CD 34 hematopoietic stem cell in vitro difficult amplified limitations; once again, a recent study found that, CD 34 the - AC 133 the transfection efficiency cells than CD 34 CD 38 - 4 times, cells in the hematopoietic stem cell gene therapy will have an important significance. Therefore, by the Lin - CD 34 - and Lin - CD 34 < sup> compare the two groups are very similar cell gene expression, cell specificity to identify Lin - CD 34 - expression may be related to traits associated genes, will provide a theoretical basis for its clinical application. On the basis of the establishment of a two-step method a negative screening Lin - CD 34 - cell using suppression subtractive hybridization (the suppression a subtractive hybridization SSH) to build of the Lin - CD 34 - cells with Lin - CD 34 cells subtractive cDNA library of differentially expressed genes. Built by subtractive cDNA library the selected clone 590, arbitrarily selected 60 positive clones were sequenced by PCR and sequencing results Genbank sequence database homology comparison. The results of 60 clones of EST sequences representing 16 genes, including four new genes EST 12 for the known gene EST BTF3, ZASP, MAPK6, Humanin gene. Studies have shown that the anti-apoptotic signal in the process of self-renewal signaling pathway may be an important part, therefore, subsequent experiments selected from the library, with the anti-apoptotic effects have been found in the nervous system Humanin depth study . Humanin obtained recently from the cerebral cortex of patients with familial Alzheimer's disease (Familial Alzheimer's disease, FAD) gene, which can specifically inhibit a variety of FAD genes, such as APP, PS-1, PS-2, etc., causing the neural apoptosis. Although the full-length transcripts in Humanin the 1,567 bp, but only 75bp, its coding frame encoding a 24 amino acid polypeptide. Further research found Humanin neuronal apoptosis induced by other factors that cause FAD also has a protective effect, it also helps mice impaired learning and memory function recovery. The the subject of Humanin gene research is mainly focused on two aspects: First the coding gene positioning Humanin, Humanin by nuclear-encoded or encoded by the mitochondrial? Second, Humanin the non-nervous system, such as the hematopoietic system, whether the same resistance to apoptosis, the mechanism how? Preliminary view that Hurnanin encoded by the mitochondrial through experimental evidence provided by the the Hurnanin sequence homology analysis and RTPCR and in situ hybridization. However, of Humanin the 24 amino acid sequence is the the standard codon translation obtained using mammalian nucleus and its gene encoded by mitochondrial contradictory. Proof of an article recently published in NatUre, and the mitochondrial codon to translate the original HUmhon sequence obtained fewer C-terminal three amino acids of the polypeptide, and still have the same anti-apoptotic role. So the source of Humanin need further experimental evidence. In this experimental study, first of all the prokaryotic expression Hurnanin peptide immunization of rabbits, and preparation of its polyclonal antibody. Cytochemical methods detect infected rabbits, Humanin located in the cytoplasm and nucleolus of HeLa cells. Nucleolar ribosomal RNA synthesis and assembly places, positioning Humanin this role is unclear. Speculated Humamn polypeptide C-terminal 2 with a positively charged arginine may be involved in this positioning, because the nuclear / nucleolar localization signal (nuclear acleolar localization signal, NLSMOS) is often rich in arginine. Subsequent yeast two-hybrid technology screening to possible molecular interactions with Humanin exist, which features a clear main four categories: one is the chaperones, such as HSP40, TCP; transcriptional regulatory factor, such as JAB, erm; a A class in points in the respiratory chain, such as cytochrome. Oxidase subunit * FO subunit of ATP synthase; class metabolism-related molecules, such as N-ethyl phenol glucosamine transferase. The study verified the Humanin, and JABI and the interaction of HSP40. The Humanin with JABI interaction with AP-l reporter gene experiments, the results show that inhibit the transcriptional activity of the AP-l Humanin through the interaction with JAB, joined JAB antisense oligonucleotide inhibition is released. Vivo HSP40 assist HSP70 play a role, so we studied the heat stress, Humanin the HSP70 colocalization, from the side to reflect Humanin with HSP40. In untreated HeLa cells, HSP70 localized in the cytoplasm, and low expression levels; 42aC heat shock treatment Zhr of expression of HSP70 upregulation and shift in the nucleolus, this time Htnnanin still localized in the nucleoli; thermal the shock treatment 37oC recovery 1,3 hr Humanin of HSP70 simultaneously removed the nucleoli Htunanin mainly located in the cell membrane and membrane, and HSP70 mainly

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