| Objectives:HE histochemical staining to analyse the evolution of morphological features of Wilms tumor in the case group; identifying gene and genotype polymorphismsex of DNMT1at rs16999593and rs75616428sites between normal and cases; immunohistochemical method study staining characters of DNMT1(1-120aa region), DMAP1and DNMT3a between tumors and normal kidney tissues; analysis of loss of heterozygosity of D1S1469, D1S1676,D16S3O5O, D16S303located at1p/16q between clinical III-IX and I-II.Methods:For this study, tumor and normal Paraffin-embedded kidney tissues are supplied by the Department of Pathology, Tianjin Children’s Hospital.1Hematoxylin-eosin histochemical staining for tumor and normal kidney tissue.2Genomic DNA extraction and agarose gel electrophoresis analysis of tumor and normal kidney tissue.3Polym erase chain reaction amplifying method applying for target region of DNMT1(NT011,295.1110291060-10291262) as also as micro-satellite loci D1S1469, D1S1676,D16S3050and D16S303.4sequencing and restriction enzymatic method analyzing amplified regions; Statistics analysis using SHEsis software with Hardy-Weinberg genetic equilibrium genotype frequencies, allele frequencies, linkage disequilibrium and haploid analysis, linkage disequilibrium was detected by the D’value and r2values to analyze, according to the polymorphisms of rs16999593(NT011295.11:10,291,181) and rs75616428(NT011295.11:10,291,113).5Immunohistochemical detection of DNMT1(1a.a-148a.a), DMAP1, DNMT3a, staining degree of blast, epithelial and stromal tissues in tumor as also as normal blood vessels, glomerular and tubular components were analyzed, analysis of variance statistics was applied according to the staining of each protein between two groups.6polyacrylamide gel electrophoresis-silver staining method is applied for detection of loss of heterozygosity (LOH) at four microsatellite sites; analysis of variance statistical analysis between clinical III-V and I-II. Results:1HE staining showed that tumor tissue contains a variety of blast, epithelial and stromal tissues, vascular component were missing in blast tissue with a small amount emerging in epithelial components; in general, compared with normal kidney tissues, blood vessels contained within tumor tissue is significantly less.2Compared with normal tissue, C/T, T/T gene frequency and genotype frequency at rs16999593site was significantly higher than normal (P<0.05), from the point of view, this polymorphism may be a predispose factor for tumor genesis.3Immunohistochemistry analysis showed that those three proteins mainly stained in nucleus, if structures are considered, it shows the blast and epithelial tissue in tumor are strongly stained (80%above); but in normal tissues the positive ratio in tubular, glomerular and vessel were90%,60%and35%respectively. But generally analyzed, no significant was observed between two groups for the staining of the three proteins, by configuring all cells in tumor and normal tissue.4The loss of heterozygosity analysis showed a higher significant was detected at D16S303between clinical I-II and III-V (P<0.1).Conclusion:WT is the embryonal tumors that originated in metanephric tissue and contains a variety of tissue components; although our study showed a significantly high ratio for C/T and C/T genetype, compared with normal tissues, it seemed that this polymorphism can’t led to structure change of DNMT1. However, the loss of heterozygosity at D16S303site can be supplied as a simple and stable detection method which may benefit for prognosis of WT patients. |
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