Fluorescence Quantitative Characterization Of Chromosome3D-spatial Organization

Many studies have concluded that the spatial organization of chromosomes in theinterphase nucleus is not random. These observations suggest that a link may existbetween the spatial organization of genomes and fundamental biological processes suchas genome reprogramming, gene expression, and differentiation. More and morechromosome aberrations have been demonstrated that they are related to cancers whichare always serious threats to human health. Until now, people usually use chemotherapyto treat cancer. However, chemotherapy not only kills cancer cells but also induces someadverse effects on the remaining cells. These adverse effects may include the change ofchromosome spatial organization, which may influence any other biological processesin cells.3D-fluorescence labeling technique and3D-fluorescence microscopic imagingtechnique have been widely used to study the structure and spatial organization ofchromosome.First, the methods involved in this dissertation were improved. The axial intensityloss of fluorescence in two-photon microscopic images of mouse oocyte chromosomeswas compensated using an intensity decay function, which could minimize the impacton chromosome’s volume calculation. In addition, a method for simultaneouslydelineating multiple targets in3D-FISH using limited channels, lasers, andfluorochromes was presented. It might open a new way for simultaneous delineation ofnumerous different targets that were defined within a nucleus depending onmorphologic parameters instead of the high configuration of scientific equipment.Moreover, the structure and spatial organization of chromosome in5-Azacytidine(5-AzaC) treated mouse oocyte in vitro were quantitatively analyzed using3D-fluorescence staining technique of whole chromosome and3D two-photonfluorescence microscopy. The present results indicated that chromosomes could regulateearly stage apoptosis and promote in vitro maturation of5-AzaC treated mouse oocyteby condensing themselves. And the instability of spatial organization of chromosomescould also induce early stage apoptosis of5-AzaC treated mouse oocyte.For further analysis, the spatial organization of chromosome territorys (CTs)8and21in mononuclear cells of patient with acute myeloid leukemia (AML) M2 t(8;21)(q22;q22) were quantitatively analyzed using3D-FISH and3D confocalmicroscopy. A hypothesis was proposed to predict the disease progression in this kind ofleukemia according to the spatial organization of chromosome. And the accuracy of thishypothesis was verified by a clinical example. In addition, the present study provided anevidence for the disputed pattern of chromosome translocation. The results indicatedthat the proximity of chromosomes was a prerequisite of chromosome translocation. Wealso indicated that chemotherapy not only killed the cancer cells but also influenced thespatial organizations of CTs. The chromosome proximity cells were neglected in clinicbut were found in this dissertation to be working as an important role in human bodyjust because the chromosomes in them have specific spatial organization. Furthermore,the data in our paper provided some degree of support for the fungibilty betweenmarrow and peripheral blood in clinical examination. This may distinctly relieve thepain of patient, when experiencing the bone marrow puncture.