| BackgroundFollicular thyroid carcinoma (FTC) is one of the most common malignant tumors of the thyroid, it accounts for about 10%-30% of differentiated thyroid cancer, but its mortality accounts for over 40% of the total mortality of the thyroid cancer. Fine needle aspiration cytology and intraoperative rapid frozen section are the most effective ways to distinguish between benign and malignant thyroid nodules, however, there are still some difficulties in distinguishing thyroid follicular carcinoma from adenoma. FTC is prone to occur hematogenous metastasis, the metastasis to the lungs, bones and brain and so on, happens in the early stage through the blood-channels; when the metastasis of FTC doesn't happen yet, FTC can be diagnosed only if capsule and vascular invasion were found on the surgical resection specimens.So the rate of misdiagnosis is high, the prognosis is not optimistic, and it seriously endangers people's health. Therefore,it is important to investigate the molecular mechanisms of FTC's occurrence and development,to screening FTC-related marker proteins for FTC's early diagnosis and treatment and improving its prognosis.The thyroid-specific transcription factor PAX8 and peroxisome proliferator-activated receptor y (PPARy) fusion gene which is named PPFP fusion gene and has been found in recent years, is an oncogene that is found closely related to follicular thyroid cancer's occurrence and development. Many studies have confirmed PPFP's positive expression rate in FTC was significantly higher than in follicular adenoma, but there are no expressions of PPFP in papillary carcinoma, oncocytic tumors, undifferentiated carcinoma. So considering PPFP fusion gene's presence as a sign of thyroid malignancy, the fusion gene may be useful to FTC's differential diagnosis and treatment. And it is gradually becoming a new entry point and focal point on researching the pathogenesis of follicular thyroid carcinoma. But the specific role and its mechanism of PPFP gene in FTC's occurrence and development process are not clear,it is worth further study. Objective:To construct the recombinant lentiviral vector containing human PPFP gene,to establish PPFP gene's stable expression cell line,and to lay the foundation for further exploring the effects and its mechanism of PPFP gene on biological characteristics in human normal thyroid cells.Methods:The PAX8 gene was isolated and amplified by PCR technique from PAX8-pOTB7 plasmid, the PPARy gene was isolated and amplified by PCR technique from PPARy-pCMV-SPORT6 plasmid. Then the two genes were directional united and this united gene was subcloned into the eukaryotic expression plasmid of pEGFP-C1 vector, to generate the eukaryotic expression recombinant vector, pEGFP-C1-PAX8/PPARy. Then the recombinant vector pEGFP-C1-PAX8/PPARy was transformed in E.coli DH5a, and the correct PPFP gene was confirmed by PCR, sequencing, analysis and contrast. Then the PPFP gene was isolated and amplified by PCR technique from PEGFP-C1-PPFP plasmid which was constructed before. And the gene was subcloned into the expression plasmid of lentiviral vector, pGC-FU(containing Flag gene), to generate the lentiviral expression vector, pGC-FU-PPFP.The correct PPFP gene was confirmed by PCR, sequencing, analysis and contrast. Recombinant lentivirus which containing PPFP gene were produced by 293T cells following the co-transfection of pGC-FU-PPFP and packaging plasmids -pHelper1.0 and pHelper2.0. The titer of virus was measured after collecting and concentrating the viral supernatant. The target cell SV40 large T antigen immortalized human normal thyroid cells Nthy-ori 3-1 was infected by appropriate amount of recombinant lentivirus that containing PPFP gene. To observe the infection efficiency. To select the cells infected with satisfactory result and to detect PPFP gene's expressions on mRNA and protein levels with RT-PCR and Western blot methods for several times. So the establishment of PPFP gene's stable expression cell line can be confirmed. Results:(1)Gene fragments PAX8-1-7, PAX8-9 and PPARy-1-6 were successfully acquired by PCR, PAX8 and PPARy were successfully directional united and this united gene was subcloned into the eukaryotic expression plasmid of pEGFP-C1 vector, to generate the eukaryotic expression recombinant vector, pEGFP-C1-PAX8/PPARy. (2) The PPFP gene was successfully isolated and amplified by PCR technique from PEGFP-C1-PPFP plasmid which was constructed before. And the gene was successfully subcloned into the expression plasmid of lentiviral vector, pGC-FU(containing Flag gene), to generate the lentiviral expression vector, pGC-FU-PPFP. Recombinant lentivirus which containing PPFP gene were produced by 293T cells following the co-transfection of pGC-FU-PPFP and packaging plasmids -pHelper1.0 and pHelper2.0,and the titer of the acquired recombinant lentivirus reached 3.5×107TU/ml. (3)The target cell human normal thyroid cells Nthy-ori 3-1 was infected by appropriate amount of recombinant lentivirus that containing PPFP gene and blank lentivirus respectively. The infection efficiency was high, it was more than 90%. the expressions of PPFP fusion gene on the levels of mRNA and protein were successfully detected by RT-PCR and Western blot.So the PPFP gene's stable expression cell line have been established. This laid the foundation for further studying PPFP gene's function and its mechanism.Conclusion:(1) The PAX8 and PPARy fusion gene (PPFP gene) was successfully cloned,and the recombinant lentiviral expression vector containing PPFP gene was also successfully established.(2) Lentiviral vector is a ideal gene transfer vector,it can efficiently transfer PPFP gene to human normal thyroid cell Nthy-ori 3-1,and the expression of PPFP gene is persistent and stable.Objective:To investigate the effects of PPFP gene's transfection on the biological characteristics of human normal thyroid cells Nthy-ori 3-1, to clarify PPFP gene's action on the FTC's occurrence and development process.Methods:With PPFP gene's stable expression cell line Nthy-ori 3-1PPFP, blank lentivirus infection cell line Nthy-ori 3-1Vector and uninfected cell line Nthy-ori 3-1 as the research object, to detect cell proliferation with MTT method, to detect the number of colony-forming with plate colony forming assay, to detect colony-forming efficiency with soft-agar colony formation assay, to detect migration and movement capacity with scratch healing experiment and to observe apoptosis and cell cycle with flow cytometer. So to observe the changes of biological characteristics before and after PPFP gene's transfection in Nthy-ori 3-1.Results:PPFP gene transfection not only significantly enhanced or increased Nthy-ori 3-1 cell's ability of cell proliferation, number of colony-forming plate, soft-agar colony formation rate, parking non-dependent growth, migration and movement capacity, but also speeded up the Nthy-ori 3-1 cell cycle progression, decreased G0/G1 phase cells'number significantly, substantial increased S phase and G2/M phase cells'number and decreased apoptosis rate significantly.Conclusion:PPFP gene transfection not only inhibited the apoptosis of human normal thyroid cells Nthy-ori 3-1 significantly,but also enhanced Nthy-ori 3-1 cell's ability of cell proliferation, migration and movement capacity significantly, this suggests that the gene may play a key role in the malignant transformation of follicular thyroid carcinoma.Objective:To further explore the specific mechanisms of PPFP gene's tumorigenic action, aiming at discovering PPFP gene's dominated and regulatory proteins, providing clues to looking for FTC's marker proteins or new drug target proteins.Methods:To investigate the changes of protein components in Nthy-ori 3-1 cells before and after PPFP gene transfection with proteomics technology.(1) With Nthy-ori 3-1PPFP cell line, Nthy-ori 3-1Vector cell line, and Nthy-ori 3-1 cell line as the research object, to created two-dimensional gel electrophoresis(2-DE) maps of three groups' cell total protein.(2) To analyze and compare the two-dimensional electrophoresis maps of the 3 groups with PDQuest software to search for differentially expressed protein spots. In these different protein points,38 differential protein spots between Nthy-ori 3-1PPFP cell line and the other two groups were chosen as candidate protein spots whose differences were more than twice in the expressions of protein content.(3) These differentially expressed candidate proteins were identified by MALDI-TOF-MS mass spectrometry analysis.(4) Select some of the protein spots,to detect the expression levels of these proteins in the 3 groups of cells.Results:(1) Successfully established three groups' cell total protein two-dimensional gel electrophoresis maps.(2) By using PDQuest software, it was found each 2-DE map contained hundreds of protein spots, most of the protein spots were consistent in the location and abundance.(2) Comparing Nthy-ori 3-1 with Nthy-ori 3-1Vector cell line,there were no obvious differences between them. But comparing Nthy-ori 3-1PPFP with the other two groups, there were some obviously different protein points.(3) 38 differential protein spots whose differences were more than twice in the expressions of protein content between Nthy-ori 3-1PPFP cell line and the other two groups were found by PDQuest software. Combined with database search, MALDI-TOF-MS mass spectrometry identified a total of 28 different protein spots. Of all the identified proteins, compared with the other two groups,19 proteins were up-regulated in Nthy-ori 3-1PPFP group cell, whereas 9 proteins were down-regulated in Nthy-ori 3-1PPFP group cell.(4) 5 proteins (Prohibiting Galectin-1,Cytokeratin 8,Cytokeratin19,HSP27) which were considered to be more closely connected to FTC,with higher scores, greater coverages, were selectively further analyzed by Western blotting to validate the results of 2-DE. The results showed that compared to Nthy-ori 3-1, Nthy-ori 3-1Vector cells, Prohibitin protein was down-regulated in Nthy-ori 3-1PPFP cell, whereas Galectin-1,Cytokeratin 8,Cytokeratin19,HSP27 proteins were up-regulated in Nthy-ori 3-1PPFP cell.The results were fully consistent with the results of proteomics research.Conclusion:(1)28 differentially expressed proteins were screened out by combination using of 2-DE and mass spectrometry techniques between PPFP gene transfer group and the other two control groups, and some differentially expressed proteins' levels were verified by Western blot, the results were fully consistent with proteomics research, and this confirmed the reliability of proteomic techniques.(2) These differentially expressed proteins may be PPFP gene's dominated and regulatory proteins,and may be closely related to PPFP gene's effects of significantly increasing proliferation, enhancing migration and movement capacity. The findings provided clues to looking for FTC's marker proteins or new drug target proteins, and laid a foundation for further clarifying the molecular mechanisms of FTC's occurrence and development. |
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