The Regulation Of Thyroid Transcription Factors On Thyroid Function Of Thyroid Follicles

Objective To verify the mechanism of the thyroid transcription factors in the regulation of thyroid function, we constructed the structure of thyroid follicles and then tested the changes of thyroid hormones through enhancing the expression of thyroid transcription factors(TTF-1、PAX-8)by lentivirus over-expression vectors.Methods 1.The logarithmic phase of Nthy-ori-3-1 human thyroid follicle cells were cultured as study objects and cultured in Transwell(Corningò3450) to construct the in vitro thyroid follicles model after the cells fusion. The model was categorized as the experimental group. The cells were cultured in six well plates were categorized as the control group. The cells were cultured 36 hours after the cells fusion with suitable concentration of iodide、1m IU/L TSH. The cellular cultured medium were collected for detecting the concentration of FT3、FT4、Tg using chemiluminescence immunoassay and the concentration of iodide using As-Ce Catalytic Chromatographer. 2.The TTF-1 and PAX-8 human gene sequences were designed accoding to data in Gen Bank and amplified by PCR technique. The segments were digested by restrictive enzyme(Eco RI and Bam HI) and connected to p LVX-puro, then it transformed competent cells E.coli. Using AMP resistance and blue/white color screening, we selected the positive colonies of transformed cells. The recombinant plasmids were confirmed by sequencing. The production of lentivirus were obtained by PEI to contransfect 293 T cells. The supermatant transfected the thyroid follicles after titer. 3. The two-dimensional models were transfected by r AAV-hr GFP 、 restruction p LVX-puro-TTF-1-HA、p LVX-puro-PAX-8-Myc.The cellular cultured medium were collected to detect the concentration of FT3、FT4、Tg and iodide.Results 1. The constructed two-dimensional models had the higher levels of the synthesis of FT3、FT4 and iodide gathering. 2.After the lentivirus transfected thyroid follicles, the levels of thyroid hormones and iodide gathering were changed due to overexpression of thyroid transcription factors. 3. The synthesis of thyroid hormones and the gathering of iodide that over-expressed TTF-1 were more than that of PAX-8.Conclusion 1. The two-dimensional models could simulate the structure of thyroid follicles in vitro model. The model has the function of synthesis of thyroid hormones and gather of iodide. 2.By the two- dimensional models,we confirmed that there were FT3 and FT4 in thyroid follicle. 3.Over-expression of TTF-1and PAX-8 could up-regulation the synthesis of thyroid hormones. The synthesis of thyroid hormones were higher in the models with over-expression of TTF-1.