Study On Molecular Diagnostic Markers In Follicular Tumors And Tumor-like Lesions Of Thyroid

Objective To study expression of Ret, Mucin-1 and Galectin-3 proteins as well as thyroperoxidase (TPO) mRNA and the sodium iodide symporter (NIS) mRNA in tumors and tumor-like lesions of thyroid, looking for valuable diagnostic markers to distinguish the benign lesions from the malignant tumors was carried out in order to enrich the diagnostic pathology in being. The related elements that connected to diagnostic markers and possible factors in thyroid tumour recurrence were analysed in order to comprehend the relationship between thyroid clinical pathological indexes and thyroid tumors.Methods 1. By means of immunohistochemical streptavidin/peroxidase (SP) technique, we selected the 212 cases of tumors and tumor-like lesions of thyroid gathered from past 10 years to detect the expression of Ret, Mucin-1 and Galectin-3 proteins, 132 among them were detected with TTF-1.18 cases of thyroid sample from surgical procedure were embedded with Lowicryl K4M low temperature embedding for immuno-electron microscopy to locate the thyroglobulin and above four markers . Meanwhile, some samples were managed with routine transmission and scanning electron microscopic techniques in order to observe the association between morphological changes of thyreocytes from various pathological thyroid and Ret, Mucin-1, Galectin-3, TTF-1 genes' in histological and ultrastructural level. 2. Abundance of the sodium iodide symporter mRNA and thyroperoxidase mRNA were detected with reverse transciption-polymerase chain reaction (RT-PCR) technique in the cases of 55 thyroid surgically resected samples. The integral of apex areas in TPO and -actin electrophoresis pieces was determined so that ratio of TPO/ -actin can be calculated which represented relative level of abundance of thyroperoxidase mRNA . 3. On the basis of TPO cDNA amplified with RT-PCR technique, the fragment was cloned to pMD18-T vector so as to constitute plasmid of pSPT19-TPO. pSPT19-TPO was separately cut by Hind HI and BamH I enzyme to form the linearization of expression plasmia. TPO cRNA sense and antisense probes were labeled with digxigenin in vivo with SP6 and T7 RNA polymerase. 10 cases of thyroid samples were tested TPO mRNA with in situ hybridization histochemistry technique. 4. To observe the relationship between the expression of Ret, Mucin-1, Galectin-3 proteins and clinic pathological indexes in tumors and tumor-like lesions of thyroid and analyse the biological behavior of them on the basis of patients' following up.143 cases of patients' clinical history was analysed. 5. Based on Stata7.0 software package, the statistical analysis of our research data was performed. Firstly, Fisher' s exact test and Bonferroni alpha correction method were used to compare positive rates between groups. Secondly, analysis of the correlation between groups was made by Spearman rank correlation.Thirdly, the quantitative analysis of TPO were made by one-way analysis-of-variance (ANOVA) models and comparing two of them with a Scheffe multiple-comparison test. Fourthly, based on analysis with one explanatory variable, multiple stepwise logistic regress analysis (stepwise backward) was performed, not only in analysis of the relation between the expression of Ret, Mucl, Gal-3 and clinical pathology characteristic, but also in the comparison of clinical pathology characteristic between malignant and benign changess. Fifthly, one way analysis and Cox proportional hazard model with stepwise backward method were used in turn in the fields of survival.Results (Part 1) Three markers were determined in 212 cases. The results were as follows. There were significant difference between benign and malignant lesions in the expression of three proteins ( P=0.000) . Ret The positive rate of malignant groups was higher than that of benign ones. The immunohistochemical staining of Ret produced cytoplasmic expression where the benign lesions (nodular goiter and adenomas) were weak positive reaction and moderate staining appear by chance, but in papillary carcinomas mostly showed with moderate and stro...