Interleukin-10Gene Polymorphism And Its Expression In Acute Myeloid Leukemia

Chapter I To establish a modified phenol-chloroform extraction of whole blood genomic DNAObjectives To establish a fast method of extracting high quality genomic DNA from whole blood, through the improvement of the traditional phenol-chloroform extraction of DNA analysis. Methods Extract genomic DNA from whole blood by applying Modified phenol-chloroform method, the classical phenol-chloroform method and Wizard (?) genomic DNA purification kit box method. Analyze DNA samples by absorption spectrometry and agarose gel electrophoresis and PCR amplification of DNA-methyltransferase3A gene fragment. Results Modified phenol-chloroform method, classic phenol-chloroform method and Wizard(?) genomic DNA purification kit extraction of genomic DNA yield, respectively (27.2±2.6) μg/ml of whole blood,(21.4±2.7)μg/ml of whole blood and (32.1±8.4)μg/ml of whole blood; purity A260/A280were1.84±0.03,1.75±0.12and1.67±0.25; extraction time were1.5±0.3h,16.5±1.0h and1.8±0.8h. Modified phenol-chloroform method with DNA extracted by the other two methods on the yield and purity, the differences were statistically significant(P<0.05);extraction time, modified phenol-chloroform method with the classic phenol-chloro imitation method difference was statistically significant(P<0.05), with the Wizard(?)genomic DNA purification kit extraction time difference not statistically significant (P>0.05). Three ways to extract genomic DNA gel electrophoresis bands, PCR amplification of the purpose of bands able to meet the analysis requirements of gene polymorphism. Conclusions Modified phenol-chloroform method is a fast method to extract high-quality genomic DNA from whole blood can be used for gene polymorphism analysis, suitable for large-scale population genomics research. Chapter Ⅱ Interleukin-10gene polymorphism and its expression in acute myeloid leukemiaObjectives To study of interleukin-10gene and its expression in acute myeloid leukemia.Methods Restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) method seized115cases of acute myeloid leukemia (AML) patients and137healthy control population of IL-10-819,-592locus single-nucleotide polymorphisms (SNP), analysis of the genotype and allele. And real-time quantitative PCR detected54cases of onset of AML patients and normal bone marrow in the control group IL-10mRNA expression, the joint genotype for statistical analysis. Results IL-10-819,-592genotype distribution in Hardy-Weinberg equilibrium. Comparison of115cases of AML patients and137cases of healthy human IL-10-819T/Cgenotype,found that of AML group-819AA genotype frequency than the healthy control group increased (59.1%vs40.9%), and in AML-819A allele frequency than the healthy control group increased (75.6%vs63.9%), statistical analysis showed that AML patients and healthy control group-819T/C genotype distribution was significantly different (P<0.05), and AML patients and healthy control group-592A/C gene distribution of the same significant difference (P<0.05). AML patients and healthy control group, the haploid TA in the frequency of AML patients than in healthy control group (75.6%vs63.9%), the difference was statistically significant (P<0.05), and two groups of IL-10three gene type distribution of the proportion of significant differences (P <0.05). IL-10mRNA expression compared with normal bone marrow in the control group was significantly increased (7.78×10-3vs2.43×10-3, P <0.05), and with Chemotherapy in AML IL-10mRNA expression levels compared with the early novo AML group decreased significantly (3.64×10-3vs7.78×10-3, P<0.05). Further analysis of the relationship between IL-10genotype, haploid with IL-10mRNA expression, found both in the initial issuance of the AML patients or normal bone marrow in the control group, the TA/TA genotype of IL-10mRNA expression levels of the lowest, CC/CC genotype of IL-10mRNA expression levels of the three genotypes of IL-10mRNA expression was significantly different (P <0.05).Conclusions1.IL-10-819,-592gene polymorphism in AML incidence,-819T,-592A allele may be susceptible to AML.2.IL-10-819,-592locus of the haploid analysis found that IL-10gene exists only in the TA and CC haploid, TA/TA, TA/CC, the CC/CC genotypes. susceptibility the haploid TA and genotypes TA/TA and the Hunan Han population in AML.3, IL-10gene polymorphism and IL-10mRNA expression analysis showed that IL-10gene polymorphism may affect the AML patients, IL-10mRNA expression.