Analysis Of Human Telomere Length At The Single-Chromosome Level By High-Sensitivity Flow Cytometry

Telomeres are complexes of DNA tandem repeats and telomere-binding proteins at the ends of chromosomes.By protecting chromosome from degradation,fusion,and rearrangement,telomere can maintain chromosome integrity and stability.Since DNA polymerase cannot completely replicate lagging-strand DNA during DNA replication,sequence loss occurs at the end of the chromosome.During the normal cellular aging process,telomeres eventually shorten after each cell division.When telomeres gradually shorten,the function of telomere is damaged and then cells with dysfunction telomeres are aging.Once telomeres shorten continuously and the length is less than 3 kb,a large scale of cells will die and the individual will be senescent or premature aging.At this time,telomerase will be activated in a few cells and these cells transform into cancer cells.Since human telomeres are intrinsic heterogeneous and the short telomeres are most important in leading to overall genomic instability and triggring senescence and cancerization,the analysis of telomere length at the single chromosome level is fundamental importance to reveal the individual difference that would be otherwise averaged out in ensemble measurements.Meanwhile,there are great differences in telomere length among species.The length of human telomeres(0.5-18 kb)is much smaller than that used in laboratory animal models(25-150 kb).It is an urgent need to develop a simple and high-throughout method for the accurate analysis of human telomere length and abundance of short telomeres at the single-chromosome level.The most commonly used techniques for telomere analysis are terminal restriction fragmentation(TRF),quantitative PCR(qPCR),STELA,quantitative fluorescence in situ hybridization(Q-FISH)and Flow-FISH.Most of them provide mean telomere length values per cell or per sample,and are thus difficult to reveal the abundance of short telomeres.In our laboratory,we have built a high sensitivity flow cytometer(HSFCM),which was 2-3 order of magnitudes more sensitive than the commercial flow cytometers.In this dissertation,we applied HSFCM to develop a sensitive,rapid and quantitative method for the multiparameter analysis of human telomere length at the single-chromosome level.This dissertation consists of the following seven sections:Chapter one introduces the telomere structure and function,along with the relationship between shortened telomere and human diseases.The conventional methods for telomere length measurement are described and the high sensitivity flow cytometry is introduced.The research plan and main contents of this dissertation are proposed.Chapter two describes the method development of chromosome analysis at the single oraganelle level via HSFCM.After optimizing the isolation conditions,we developed a universal method for chromosome isolation and purification from immortal cell lines.The chromosomes were labeled with the nucleic acid dyes such as PI and Picogreen,and the monokaryotye was analyzed via HSFCM.Based on the number of chromosomes and the corresponding DNA content,theoretical simulation using Matlab was employed to predict the monokaryotype of chromosomes from HeLa cells.Meanwhile,the isolated chromosomes were observerd under the fluorescence microscopy and the relationship between fluorescence intensities of single chromosomes and their DNA contents was analyzed.Trough these approaches,we confirmed the purity and integrity of isolated chromosomes in suspension.Chapter three describes the development of a high-throughput and quantitative method for telomere length analysis at the single-chromosome level via HSFCM.By using a specific peptide nucleic acid(PNA)probe and optimizing hybridization conditions,a method for detecting telomere length via fluorescence in situ hybridization in suspension was established.The fluorescence of telomere was quantified by using three fluorescent nanoparticles with known AF488(Alexa Fluor 488)equivalents as standards to construct a calibration curve,which was then used for the conversion of the fluorescence intensity of single chromosome to telomere length.Chapter four describes the comparison between HSFCM and three conventional methods(terminal restriction fragment,quantitative PCR and quantitative fluorescence in situ hybridization).We analyzed five immortal cell lines(HeLa,A549,HEK293T SMMC-7721 and Hep G2)by HSFCM and these three conventional methods.Compared with these three conventional methods,the HSFCM method exhibits unique advantages in the rapid and quantitative measurement of telomere length of human cells.Chapter five describes the application of the as-developed HSFCM method for the telomere length analysis of lymphocyte in clinic peripheral blood samples.The abundance of short telomeres was compared between healthy donors and patients with chronic myeloid leukemia(CML)to see whether it can be used to predict the efficacy of therapeutics.Moreover,the quantity of short telomeres was compared among paients with acute leukemia,patients in different phases of CML and healthy donors to see whether it can be used as a marker for disease progression prediction in CML.Chapter six describes the analysis of telomere length of specific chromosomes.We upgraded the high sensitivity flow cytometer with dual lasers of 488 nm and 642 nm excitation wavelength.Two PNA probes specifically targeting the repeated sequence of telomere and human alpha-satellite DNA of specific chromosomes,respectively were used simultaneously.So that specific chromosomes can be identified with concurrent analysis of its telomere length.X chromosome parental homologs were found in HeLa cells.Chapter seven summarizes the content of present thesis and proposes the future directions of the present research.