Construction Of CDNA Subtractive Library For Lin～-CD₃₄～- Cells And Research On Antiapoptotic Effect Of Humanin In K562 Cells

CD₃4 is a cell-surface sialomucin-like adhesion molecule that is expressed on 1%-3% of bone marrow (BM) cells. Because the CD₃₄⁺ population appears responsible for most, if not all, of the hematopoietic activity in BM, CD₃4 has been used as a convenient positive selection marker for HSC both in research and in clinic. But recently, using two elegant animal models, human/sheep competitive engraft model and NOD/SCID mouse transplantation model, some researchers showed that there is another population of HSCs which do not express lineage commitment markers nor the CD₃4 molecule. These CD₃4 HSCs possess several characters differ from former CD₃₄⁺ HSCs. First, recipients transplanted with Lin"CD₃4 cells showed more potential in the long-term reconstitution ability, more rapid reconstitution of the immune system than CD₃₄⁺ cells transplantation; Second, in vitro, CD₃₄⁺ HSCs could be expanded greatly and give rise to a large population of CD₃₄⁺ cells; Third, CD₃₄⁺AC₍133_<sup>+ cells show a 4 times superior potential for retroviral transdction to the CD₍34_<sup>+CD₃₈⁺ cells. Comparing these two similar populations about their gene expression profiles may provide us some useful information on Lin"CD₃₄^- HSCs' manipulation and clinical application.Two-step negative selection method was developed to select for Lin"CD₃₄^- cells which lack a positive marker. Using suppression subtractive hybridization technique, cDNA subtractive library was constructed between Lin"CD₃₄⁺ and Lin"CD₃₄⁺ cells. A total of 590 clones were obtained, and after PCR screening, 60 randomly picked clones were sequenced for further analysis. Homologous analysis revealed that these 60 clones contained 12 known genes and 4 novel genes. The known genes are BTF3, ZASP, MAPK6, Humanin, et al. Among these, Humanin was selected for further research for its antiapoptotic effect.Humanin is a recently identified peptide from the occipital cortex of an Alzheimer's disease patient brain, which takes the function of suppress neuronal cell death by various familial Alzheimer's disease mutants, such as APP mutant, PS1 mutant and Aβ peptide. Humanin has a full length of 1,567bp, which contains a 75-base ORF that encodes a 24-residue peptide MAPRGFSCLLLLTSEIDLPVKRRA.Our research about Humanin is focus on two aspects. First, to make sure is Humanin encoded by nuclear or mitochondria? Second, could Humanin exert its antiapoptotic effect in other systems than neural system? And what's the mechanism underlying?Homologous analysis combined with RT-PCR and in situ hybridization, Humanin was belived to be encoded by mitochondria. But its peptide sequence is translated using mammalian nuclear standard codon, which is controversial with its mitochondric location. Recently, a paper published on Nature proved that if Humanin was translated using mammalian mitochondric codon, which give out a 3 amino acids short peptide, it still preserve its antiapoptotic effect. So, the origin of Humanin need further research.To facilitate the following research, prokaryotic expressed Humanin peptide was used to immune rabbit to obtain polyclonal antibody against it. Using immunocytochemistry, Humanin was located to cytoplasm and nucleolus hi HeLa cells. Nucleolus is a place for ribosomal RNA synthesis and ribosome assemble. Why Humanin located here is still unknown. But we suspect that two Arginines located to its C-terminal are involved in its localization to nucleolus, because nuclear/nucleolar localization signal used to rich for Arginine.Using yeast two-hybrid system, proteins interacting with Humanin were screened. They fall into four classes: First, chaperons, such as HSP40 and TCP1; Second, transcription regulation factors, such as JAB1 and erm; Third, molecules compose respiratory chain, such as cytochrome c oxidase subunit II and ATP synthase, H+ transporting, mitochondrial FO complex; Forth, metabolism related, such as N-acetylglucosaminyltransferase VI. In this research, interaction between Humanin and JAB1 or HSP40 was studi...