| Thyroid cancer is the most common malignant tumor of the endocrine system, Papillary thyroid carcinoma(PTC) is the most common type of pathological, the incidence of rising. Recently, significant attention of a wide range of researchers has been toward to relationship between sonic hedgehog signaling pathway and human diseases. However, studies of Shh pathway involved in papillary thyroid carcinoma insult has been poorly documented. The studies in this thesis focus on the Shh protein and Shh signaling pathway in PTC. Aim to clarify the effects and possible signal transduction mechanism, which identify novel potential target for the treatment of PTC.1 Expression of Shh/Gli1 in papillary thyroid carcinoma and its clinical biological significanceObjective:To define the expression of Shh/Gli1,the key elements of Hedgehog signaling pathway in papillary thyroid carcinoma(PTC) and to explore the relationship between the expression of Shh/Gli1 and clinical significance.Methods:The expression of Shh and Gli1 was examined in 142 cases of PTC tissues and corresponding adjacent-tumor thyroid tissues as control by immunohistochemistry. Analyze the relationship between the expression of Shh/Gli1 and clinical characteristics of PTC patients.Results:The positive rate of the cytoplasm of Shh expression and the nuclear of Gli1 expression was 64.1% and 47.9%,respectively. Significant difference was found between normal thyroid tissues and PTC.The research showed that the expression of Shh/Gli1 was related to the tumor size,clinical stages and lymph node metastasis, Shh was more significant in the tumor size(P<0.01) and Gli1 was more significant in lymph node metastasis(P<0.01).Conclusions:Varying expression of the main ligand Shh and transcription factor Gli1 in Hedgehog signaling pathway was found in PTC.The expression of Shh/Gli1 was related to the tumor size, clinical stage and lymph node metastasis,indicating that the aberrant activation of Shh signaling pathway plays some role in PTC. Shh/Gli1 may be indicators for prognosis and ideal targets for therapy against PTC. 2 Long-term primary culture of human papillary thyroid carcinoma cellsObjective:To investigate the primary culture method of human papillary thyroid carcinoma cells for a long term and to establish a monitoring and verification measures.Methods:Papillary thyroid carcinoma cells were isolated following routine procedures and cultured in the MEM supplemented with 10% fetal bovine serum (FBS), glutamine and 20ng/ml EGF. Thyroglobulin (Tg) and thyroperoxidase (TPO) in nutrient solution and specific antigen Tg expression of PTC cells cultured for different days were observed.Results:The PTC cells grew satisfactorily up to 45 days of incubation. Tg content in nutrient solution express the training period of a linear singular parabolic, achieves peak value(985.2μg/L) in about 14d. TPO has not been detected in nutrient solution. The thyroid globulins occurring expressed positive by immunization fluorescent dyeing.Conclusions:PTC cells cultured with present method are growing up to over 45 days. A brief, monitoring and evaluation systems of PTC cells is established. The report prompts that in gene research about 14d activity in the cells may be used more suitable. To research thyroid papillary carcinomas events and features of the mechanisms is provided a suitable substitute for tools.3 Research of gene expression of Shh gene and preliminary experiment of lentiviral vector in PTC cellsObjective:To investigate gene expression and dynamic of preliminary experiment of lentiviral vector in PTC cells for providing information to guide the further gene interference experiment.Methods:Shh and Tg mRNA expression of the primary culture method of human papillary thyroid carcinoma cells were observed with Real-time PCR and Western blotting. To explore conditions of transfection from PTC cells by using different amounts of polybrene,et al,in different times, then to test and evaluate the model.Results:Tg mRNA expression of purpose cells was satisfaction, thus purpose cells were identified to PTC cells. Expression abundance of Shh mRNA cannot satisfy demands of detecting experiment. The MOI need about 20 when to obtain 80% of the interference efficiency by the best condition for this situation was Eni.S with 5ug/ml Polybrene.Conclusions:Expression abundance of Shh mRNA cannot satisfy demands of detecting experiment, gene silencing biotechnology may not fit the further gene interference experiment; PTC cells can be infected by lentiviral vectors suggested providing information about transfection experience. 4 Construction and Identification of Lentiviral Expression Vector Carrying ShhObjective:To construct a lentiviral expression Vector Carrying Shh and obtain Shh efficient and stable expression in 293T cells.Methods:The DNA fragment of Shh coding sequence was amplified by PCR with designed primer from the plasmid of Shh,and then subcloned into pGC-FU vector with In-Fusion technique to generate the lentiviral expression vector,pGC-FU-Shh.The positive clones were screened by PCR and the correct Shh was confirmed by sequencing. Recombinant lentiviruses were produced in 293T cells following the cotransfection of pGC-FU-Shh,and packaging plasmids of pHelper 1.0 and pHelper 2.0. Shh protein expression in 293T cells was detected by Western blotting.Results:The lentiviral expression Vector Carrying Shh was successfully constructed and Shh protein expression was detected in 293T cells.Conclusions:The recombinant lentiviruse pGC-FU-Shh-GFP was successfully produced and it can establish a foundation for our study the molecular function of Shh.5 Study on the changes of PTC cell's biological properties by constructing stable Shh-overexpressing cell lineObjective:To investigate Changes in of PTC cell's biological properties by constructing stable Shh-overexpressing cell line and aim to clarify the effects and possible Shh signal transduction mechanism.Methods:PTC cells were infected with lentiviruse pGC-FU-Shh-GFP. Shh protein expression was detected. The proliferative and migratory activities of every group were detected using cell active assay(MTT) and Transwell cell migratory assay. Flow Cytometry (FCM) was performed to observe the effects of Shh overexpression on the distribution of cell cycle of PTC cells. Detecting the expressions of Shh signaling pathway related genes (RTCH,SMO,Gli1)of PTC-Shh cells.Results:The proliferation of PTC-Shh a cells was improved comparing with PTC-vector cells through MTT test and cell counting. Overexpression of Shh caused the increase of the percentage of Gl stage of PTC cell. Transwell cell migratory assay suggests that improvement of Shh expression suppress migratory activity of PTC cell.Comparing with PTC-vector cells,the expressions of mRNA of genes RTCH,SMO and Gli1 increased in PTC-Shh cells..Conclusions:To activate Shh signaling pathway in PTC cells through Shh overexpression PTC cells that is constructed through infecting and selecting. To activate Shh signaling pathway is the promoting factor and the occurrence in PTC.The report suggests that the mechanism may stimulate the growth and metastasis of the PTC. |
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