| Objective and designMany diseases can cause hyperthyroidism, of which the most common is Graves disease (GD)(account for 90%). GD is one of organ specific autoimmune diseases. It relates with inheritance, environment and immunity. Papillary thyroid carcinoma (PTC) is the most common thyroid malignancy. PTC constitutes 60 to 80% of thyroid carcinoma. As the complexity of these diseases, the tools available to clarify its regulation also need to be advanced.cDNA microarray technology is a powerful descriptive method for examing the expression profile of hundreds to thousands of genes in unison. Dr. Fodor from Affymetrix company in US firstly developed the gene chip technology in early 1990's. Since then it has been used successfully in various area of gene function research. Large-scale gene expression profiling using cDNA arrays has become one of the most fruitful methods for characterizing the physiological and pathological processes. But the research of gene chip technology in GD and PTC is scarce. Using this technique to look for differentially expressed genes in GD and PTC will be helpful for the analysis of the mechanism, clinical diagnosis and treatment of them. In this study, we have screened genes associated with GD and PTC, and verify some genes with RT-PCR and immunohistochemistry. Four sections were included in our experiments: 1) to screen the immune related genes with GD of cDNA microarray; 2) to identify the differentially expressed genes with GD by means of RT-PCR and immunohistochemistry; 3) to scan the related genes with PTC of gene chip; 4) to verify the differentially expressed genes with PTC by RT-PCR and immunohistochemistry. Methods and Results 1. to screen the immune related genes with GD of cDNA microarrayMethods: Three patients with GD are female, whose age is from 41 to 46 years old. Before operation they use antithyoid drugs to control symptom and decrease the level of thyroid hormone. Moreover they were treated with iodine for two weeks. The control tissue was collected from 2cm beside thyroid adenoma. All of the tissues were preserved in liquid nitrogen. Both mRNA from the Graves disease and the normal were reversely transcribed to cDNAs with the incorporation of fluorescentdUTP(cy-5 or cy-3) to prepare the hybridization probes. The mixed probes were hybridized to the cDNA microarray, which include 578 genes. After high-stringent washing, the cDNA microarray was scanned for the fluorescent signals and showed differences between two tissues. Results: We identified 80 differentially expressed genes between two tissues. In GD 31 genes were upregulated while 49 were downregulated. The genes are associated with cytokine and its receptor, adhesive molecule, complement, signal transducer and activator of transcription (STAT1), bone morphogenetic protein 7(BMP7) and its receptor, catalase, toll-like receptors, oncogene and so on.2. to identify the differentially expressed genes with GD by means of RT-PCR and immunohistochemistryMethods: 13 GD thyroid tissue were obtained from patients operated for it, including five men and eight women, whose age is from 33 to 54 years old. Properative use of antithyroid agents decreased the level of T3, T4 to normal. They were also treated with iodine for two weeks. 13 normal tissues were got from patients underwent thyroidectomy for thyoid adenoma. All tissues were divided into two parts, one for RT-PCR: design and synthesis the primers of STAT1, BMP7 and P -actin. Then we extracted RNA for RT-PCR and examined the band intensity of these two genes; another for immunohistochemistry: we evaluated staining intensity of STAT1 and BMP7 by Axioskop2 plus microscope and image analysis system, calculating the positive findings. Results: the expression of STAT1 in GD was higher than the control. On the opposite, the expression of BMP7 in GD was lower than the control. 1'he histological examinations showed the hyperplasia of the follicular, which is inequality of size. The buffy staining located in the plasm of acinar cells. The statistic analysis showed th... |
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