Study On Fabrication And Application Of Gene Expression Profile Microarray Of Human Leukemia K562 Cell

K562 cell is a pluripotential human erythroleukemia cell line that can be induced by a number of chemical agents such as hemin and hydroxyurea to undergo erythroid differentiation. The cell line remains an attractive model for the study of cancer chemotherapy and cell differentiation.Gene chips or DNA microarrays technology, which arises in recent years and intercrosses molecular biology, micro-electronics and other disciplines, is a powerful tool for analyzing hundreds and thousands of genes simultaneously so that it can be used to study gene expression profile in a high-throughput way. The processes of implementing this technology are: preparing a large number of cDNA fragments as probes, selection and surface treatment of solid substrate, microarray printing, sample labeling, molecular hybridization, scanning and information analysis. In this paper, it was systemically studied how DNA microarrays for gene expression profile of K562 cells were fabricated and applied to the research of gene expression changes in K562 cells induced into differentiation by agents. The main results are as follows:1. Restriction display (RD-PCR) technology was applied to prepare cDNA probes of human leukemia K562 cells for gene expression profile chips. Two kinds of new methods for cDNA probe-preparation were designed here. (1)After RD-PCR amplification, cDNA fragments were isolated by polyacrylamide gel electrophoresis (PAGE) and were displayed by nucleic acid silver staining technology. The single bands were cut out from the gel and used as second round PCR amplification. Then PCR products were purified as probes. (2)The RD-PCR products of each subgroup were cloned into T-vectors and then transferred into E coli. JM109 strain, respectively. Thus a restriction cDNA library was constructed. The clones were identified and isolated from different groups, then probes for cDNA microarray were amplified. Each group of this library contains specific cDNA, so the identification and isolation of clones were dramatically accelerated. In these way, 2628 different cDNA fragments from K562 cells before and after induced by hydroxyurea have been isolated. The main cDNA fragments were 250 ~ 750 bp long. The sequencing resultsof some cDNAs fragments showed that they were cancer related genes, cell cycle related genes, cell metabolizing pathway related genes, signal transduction and transcript regulation related genes and so on. Two new cDNAs were found and might be relate to the differentiation of K562 cell.2. The fabricating conditions of DNA microarray of gene expression profile in K562 cells were systematically studied. The results showed: (D The cost is lower, output is higher and cDNA is easy to dissolve when the cDNA probes amplified by PCR were purified with 2.5 volumes of ethanol and 1/10 volume of NaAc(pH 5.2). d) The optimum concentration of cDNA probes dissolved in 50% DMSO is 0.3 g/L. The dots of array diffuse smaller and print pins were not apt to clogged. (3) The DNA attachment rate of the glass slide surface, which was modified with aldehydation, isothiocyanic acidation or amino-group, was compared and showed that of the slides treated with isothiocyanic acidation was higher than those of the other two treatments, about 70%, and was close to those of the amino-group modified glass slides imported. But the difference among the different treatments and between the slides made in china and the slides imported is not remarkable. ? When the amino-grouped slides made by Corning company were used to print the probes on, microarray was cross-linked by 150 mJ UV-ray and dried 2 h at 80°C, the DNA attachment rate is 97%. (§) The sample volume of 15-20 uL and 27-30 pre-print numbers give a better array.3. The length of probes prepared by the technology of restriction display PCR is similar, and fit to hybridization of gene chip. When the hybridization time is from 16 h to 20 h under the condition of hybridization solution containing 25% formamide at 42V, the specificity of hybridization is high. The sensitivity of detection could be improved when universal primers labeled by fluorescence(Cy3 or Cy5) were used to label little sample according to restriction display PCR or liner PCR, and the later is a liner amplification. The effect of directly labeling total RNA is same as that of labeling mRNA. The amount of total RNA for one hybridization is 0.5-1 Oug. To an array of 1000-1500 dots/cm2, the amount of labeled sample is about l~2ug.Purifying the labeled samples with PCR purification kit can largely reduce the...