| Uterine endomerial carcinoma (UEC) is a common genycological malignancy of the female urogenital tract, though as diagnosis and treatment rises , the UEC patient in early stage, whose survival rate of 5 years rose greatly, but patients in later period and low differentiation whose survival rate only attained 5-15%, and patients with ER PR unexpression whose prognosis would be worse,whose endocrine treament would be of no avail. Endometrial cancer induced by hyperestrogenism is suppressed by progesterone via PR . Human PR has two isoforms,A and B,and several studies indicated PRB had a stronger transcriptional activating function,and the PRB:PRA ratio is abnormal in endometrial cancer,leading to a lack of normal progesterone protection against the growth-promoting effects of E2. So the lose of PRB was more related to the incidence of UEC. CpG islands are areas rich in CG dinucleotides that are found with in the promoter region of various genes. Abnormal CpG island methylation seems to be a frequent event in most malignancies, and the hypermethylation would lead to gene silence. Sasaki found that methylation of CpG island of PRB gene promoter region occured in UEC and mRNA of PRB re-expression was induced by inhibitor factor of Dnmt on several UEC cell line. The domestic reports have not been seen. In this study, the methyltion incidence of CpG islandin promo tor region of PRB gene in UEC was investigated, and on Heccl-1 UEC cell line , the expression of PRB was checked at protein level before and after the action of 5 ' -aza-C to explore furtherly whether the mechanism of PRB unexpression in UEC were related to methylation of gene or not ,and to offer experiment basis for application of demethylating agent to the clinical treatment on UEC. 2.Materials and methods2.1 Materials: 26 cases of UEC and 7 cases of normal uterine endometrial are selected from 1997-1999 paraffin-embeded samples. UEC cell line-Heccl-1 came from the research institute of tumor prevention and treatment of Shandong province.2.2 Methods2.2.1 Check of PRB gene 5'CpG3' island methylation in UECDNA was isolated from the samples scraped from paraffin-embeded sections with UNIQ-10 genomic mininum DNA extraction kit. The methylation status of the PRB gene in tumors was determined by the methylation-specific PCR (MSP) described by Herman. Briefly,genomic tumor DNA was modified by treatment with sodium bisulfite,which converted all unmethylated cytosine residues to uracil, and uracil was then converted to thymidine in the subsequent PCR,while methylated cytosine could resist this modification and maintain its original sequence. Modified genomic DNA as template, Primer set specific for methylated sequence(PRB-M) was used to amplify the promoter region of the PRB gene that incorporated a number of CpG sites.The ampification products were separted on a 3%agarose gel and visualizd by ethidium bromide staining and Uv transillumination. If 202bp specific PCR products existed, which indicated that methylation of CpG island in the promoter region of PRB gene occurred in this sample.2.2.2 Check of PRB protein on Heccl-1 cells before and after action of 5 ' -aza-CHeccl-1 cells were maintained in IMDM with 20%FCS.The cells were treated with a freshly prepared solution of 5 ' -aza-C. On day 1, different final concentration of 5 ' -aza-C in PBS were respectively added to the flask. The next day ,the medium was changed .On day 3 and 5,the cells were treated with 5 ' -aza-C two more times.On day 6,the cells were harvested and droped on slide fixed with acetone. Antibody specific for PRB was used to identify PRB expression on the slide with SP immunohistochemistry kit and DAB immunostaining. Blank contrast was established by PBS replacing first antibody. The judgement of result: compared with ground staining ,brown color stained obviously at the cell nuclear,which type of cells would be think as typical PRB expression positive ones. Counting 100 cells under different rap of HP on every dropping spot, if the number of typic...
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