| Background:Intermediate filaments are primary components of mammalian cell cytoskeleton and constitute a multigene family of proteins distinguished by their cell type-specific expression. Immunological and biochemical criteria allow us to divide it into five different types. The cytokeratin(CK), one of the intermediate filaments which comprise at least 20 differents isotypes, are predominantly expressed in epithelial cells. The cytokeratins can be put into two categories on the basis of their physical and chemical nature, I -CK ( CK9~20, acidic cytokeratin polypeptides ) and II -CK ( CK1-8, basic cytokeratin polypeptides ). CK20 is the most recently described cytokeratin isotype which isolated and characterized from mucosal epithelium of human small intestine by Moll et al (1990). A large number of immunhistochemical analysis showed that the expression of CK20 is almost entirely confined to gastric and intestinal epithelium, pancreas and bile system epithelium, cutaneousMerkel cells and several epithelial cells in patients with tumors such as blabber transitional cell carcinoma ( TCC ). Because of its restricted expression in tumor or normal tissue, CK20 has widely applied to differentiating primary or metastatic carcinoma, diagnosing benign or malignant tumor, monitoring hematogenous dissemination of perioperation and so on. Klein et al. showed that, despite the fact that malignant cells generally retain the intermediate filaments of their progenitors, the exfoliated cells of normal urine do not express the CK20 gene, but urinary specimens of TCC can be detected CK20 gene. Their findings emphasize the possibility that CK20 is a specific biomarker for detecting TCC in voided urinary specimens. But Southgate et al, made an objection to this viewpoint. They believe that not only the cells of TCC but also the cells of urothelial dysplasia or normal cells express CK20 although the number of CK20 of those cells are different. To clear the mind about the pattern of CK20 expression in cells of voided urine, we detect CK20mRNA in normal, benign and malignant cells by sensitive reverse transcriptase-polymerase chain reaction and specific DNA sequencing using CK19mRNA as control gene.Purpose:At present urinary cytology and cystoscopy are the primary methods in the diagnosis of TCC before surgery. The former has poor sensitivity and specificity, particularly in well differentiated bladder tumors, and the latter remains invasive to patients. The aim of this experiment is to find a more sensitive, specific and noninvasive detection method assesses urothelial cells form the voided urine specimens using molecular biological technology.Materials and Methods:1. Cells was obtained from the urine of 43 patients with bladder tumor according to cystoscopy before operation and 17 healthy volunteers without a history of TCC. Total RNA was immediately extracted and reversely transcribed to cDNA. PCR was processed with 2 specific primers lie in CK19 and CK20 gene. RT-PCR products were analyzed by2% agarose gel electrophoresis with ethidium bromide. Appearance of 370bp band presented positive CK20 results and 214bp to CK19.2. The random RT-PCR products were sequenced using the ABI Model 377-96 DNA sequencer by 5% PAGE, then aligned and analyzed using an Apple Macintosh computer. The final sequence was obtained by comparison between forward and reverse sequence.3. To evaluate the effect of CK20 specific RT-PCR method in detecting TCC, the results of agarose gel electrophoresis were compared with histological sections and cystoscopic examinations. The pattern of CK20 expression in 3 groups of healthy volunteers, patients of negative bladder biopsy for TCC and bladder TCC was observed and analyzed. The sequences was compared with data of human CK19 and CK20 gene bank ( www.ncbi.nim.nih.gov) to examine the specificity of this method.Results:1. Although CK19 was expressed in all of 3 groups, the positive rate of CK20 is 0% in healthy volunteers, 5.9% in patients without TCC and 88.5% in patients with...
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