【作者】 刘剑鸣；

【导师】 王志明；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 研究背景甲状腺滤泡状癌(follicular thyroid carcinoma,FTC)是最常见的甲状腺恶性肿瘤之一,约占分化型甲状腺癌的10%-30%,但其死亡率却占甲状腺癌总死亡率的40%以上。细针穿刺细胞学检查及术中快速冰冻切片是鉴别甲状腺结节良恶性的最有效方法,然而对于滤泡状癌和甲状腺腺瘤的鉴别却仍存在一定的困难。FTC易发生血行转移,早期即可通过血行播散转移至肺、骨骼和脑等部位；未转移时只有在手术切除的标本上发现包膜和血管侵犯方能确诊,因此误诊率高,预后并不乐观,严重危害人民群众健康。因此探讨FTC发生发展的分子机制,筛选FTC相关的标志蛋白,对于FTC的早期诊治和改善预后均有重要意义。甲状腺特异转录因子PAX8与过氧化物酶增殖激活受体γ(PPARy)的融合基因,即PAX8/PPARγ融合基因(PPFP基因)是近年来发现的与FTC发生、发展密切相关的致瘤基因,已有不少研究证实,PPFP在滤泡状甲状腺癌中的阳性表达率明显高于滤泡状腺瘤,而在乳头状癌、嗜酸细胞肿瘤、未分化癌中未见PPFP的表达,因此认为PPFP融合基因的出现高度预示甲状腺恶性病变,该融合基因可能会对甲状腺癌的鉴别诊断和治疗有帮助,并正逐渐成为研究FTC发病机制新的切入口和聚焦点。但PPFP基因在FTC发生发展中的具体作用及其机制尚不明了,值得进一步研究。目的：构建含PPFP基因的慢病毒表达载体,并建立PPFP基因稳定表达细胞株,为进一步探讨PPFP基因对人正常甲状腺细胞生物学特性的影响及其机制奠定基础。方法：从含有PAX8基因和PPARy基因的质粒克隆模板PAX8-pOTB7和PPARγ-pCMV-SPORT6中,利用PCR方法分别钓取目的基因PAX8和PPARγ,将PAX8和PPARγ定向连接后克隆到pEGFP-C1载体上,构建PAX8和PPARy融合基因的真核表达质粒pEGFP-C1-PPFP,并转化大肠杆菌E. coli DH5 a,再通过PCR、测序和分析比对验证PPFP基因。然后从已构建好的含PPFP基因的质粒克隆模板PEGFP-C1-PPFP中,利用PCR方法调取目的基因PPFP,将该基因克隆到慢病毒载体表达质粒pGC-FU(含Flag基因)中,得到重组的pGC-FU-PPFP,通过PCR、测序和分析比对验证PPFP基因后,将pGC-FU-PPFP质粒和包装质粒pHelper1.0、pHelper2.0共同转染人胚胎肾上皮细胞株293T细胞,获得携带PPFP基因的重组慢病毒,收集并浓缩病毒上清液,测定重组慢病毒的滴度。再以携带PPFP基因的重组慢病毒感染目的细胞SV40大T抗原永生化人正常甲状腺细胞系Nthy-ori 3-1,观察感染效率。选择感染效率满意的细胞通过RT-PCR和Western blot多次检测PPFP的表达,以确定PPFP基因稳定表达细胞株的建立。结果：(1)PCR方法成功获得目的基因片段PAX8-1-7, PAX8-9及PPARy-1-6,并成功将PAX8和PPARy拼接,构建了PAX8与PPARy的融合基因的重组真核表达质粒pEGFP-C1-PAX8/PPARγ。(2)以重组真核表达质粒pEGFP-C1-PAX8/PPARγ为模板,PCR方法成功获得目的基因PPFP,并成功构建PPFP基因的重组慢病毒表达质粒pGC-FU-PPFP,以该质粒与包装质粒共转染293T细胞,成功获得携带PPFP基因的高滴度的重组慢病毒,病毒滴度达3.5×107TU/mL。(3)以携带PPFP基因的重组慢病毒和空白慢病毒分别感染人正常甲状腺细胞Nthy-ori 3-1,感染效率高,大于90%,以RT-PCR和Western blot检测证实PPFP基因在mRNA和蛋白水平顺利表达,PPFP基因稳定表达细胞株成功建立,为进一步研究PPFP基因的功能及其作用机制奠定了基础。结论：(1)成功克隆了人PAX8与PPARy的融合基因(PPFP基因),并构建了PPFP基因的重组慢病毒载体；(2)慢病毒载体是理想的基因转移载体,可以高效的将PPFP基因转染至人正常甲状腺细胞Nthy-ori 3-1,转染后PPFP基因可持续稳定表达。目的：探讨PPFP基因转染对人正常甲状腺细胞Nthy-ori 3-1生物学特性的影响,以明确PPFP基因在FTC发生发展过程中的作用。方法：以PPFP基因稳定表达细胞株Nthy-ori3-1PPFP、空白慢病毒感染细胞株Nthy-ori3-1Vector以及未感染细胞株Nthy-ori 3-1为研究对象,通过MTT检测各组细胞增殖能力、平板克隆形成实验检测各组细胞体外克隆形成能力、软琼脂集落形成实验检测各组细胞停泊非依赖性生长、划痕愈合实验观察各组细胞体外迁徙运动能力以及流式细胞仪检测细胞凋亡和细胞周期,观察PPFP基因转染前后Nthy-ori 3-1细胞生物学特性的改变。结果：(1)PPFP基因转染可促进人甲状腺细胞Nthy-ori 3-1增殖。(2)PPFP基因转染可提高Nthy-ori 3-1细胞体外克隆形成能力。(3)PPFP基因转染可促进Nthy-ori 3-1细胞的停泊非依赖性生长。(4)PPFP基因转染可增强Nthy-ori 3-1细胞体外迁徙运动能力。(5)PPFP基因转染可使Nthy-ori 3-1细胞周期进程加快,细胞G0/G1期细胞减少,S期及G2/M期细胞增加,并大大降低Nthy-ori 3-1细胞的凋亡率。结论：PPFP基因转染可显著抑制人正常甲状腺细胞Nthy-ori 3-1凋亡,并显著促进人正常甲状腺细胞Nthy-ori 3-1的增殖能力和迁徙运动能力,提示该基因可能在滤泡状甲状腺癌恶性转化中起关键性作用。目的：探讨PPFP基因发挥致瘤作用的具体机制,筛选PPFP基因调控蛋白,以期为寻找FTC的标志性蛋白或新的药物靶标蛋白提供线索。方法：以蛋白质组技术探讨转染PPFP基因前后Nthy-ori 3-1细胞蛋白质组分的改变。(1)采用2-DE技术分别制备PPFP基因稳定表达细胞株Nthy-ori3-1PPFP、空白慢病毒感染细胞株Nthy-ori3-1Vector以及未感染细胞株Nthy-ori 3-1总蛋白质的双向凝胶电泳图谱。(2)应用PDQuest软件对双向电泳图谱进行比较分析,寻找差异表达的蛋白质点。选择Nthy-ori3-1PPFP与另外两组细胞差异均在两倍以上的38个蛋白质斑点作为候选蛋白质点。(3)采用MALDI-TOF-MS质谱分析技术对这些差异表达的蛋白质进行鉴定。(4)选取部分差异蛋白质点,以Western blot检测各蛋白质在三组细胞中的表达水平。结果：(1)采用2-DE技术建立了Nthy-ori 3-1、Nthy-ori3-1Vector和Nthy-ori3-1PPFP三组细胞的双向凝胶电泳图谱。(2)应用PDQuest软件分析发现,每张2-DE图谱均有数百个蛋白质点,大多数蛋白质点在位置和丰度上是一致的；Nthy-ori 3-1和Nthy-ori3-1Vector细胞2-DE图谱的蛋白质点无明显差别,绝大部分点在位置和丰度上均一致；而Nthy-ori3-1PPFP与上述两组细胞相比,存在一些差异较为明显的点。(3) PDQuest软件共发现Nthy-ori3-1PPFP与另外两组细胞差异均在两倍以上的38个蛋白质斑点,采用MALDI-TOF-MS质谱分析技术结合数据库的搜索,共鉴定出了28个差异蛋白质点。其中,Nthy-ori3-1PPFP与另外两组相比,表达上调的点有19个,表达下调的点有9个。(4)选取其中MALDI-TOF-MS鉴定分数较高,覆盖率较大,且认为与FTC发生发展关系较为密切的5个蛋白质(Prohibitin、Galectin-1、Cytokeratin 8、Cytokeratin19、HSP27)作为验证对象,Western blot方法检测发现,与Nthy-ori 3-1、Nthy-ori3-1Vector组相比,Prohibitin在Nthy-ori3-1PPFP组细胞中表达下调,Galectin-1、Cytokeratin 8、Cytokeratin19、HSP27在Nthy-ori3-1PPFP组细胞中表达上调,这与蛋白质组学研究结果完全一致。结论：(1)采用2-DE技术结合质谱鉴定技术共筛选出PPFP基因转染组细胞较对照组细胞28个差异表达蛋白质,并对部分差异蛋白质的表达水平进行了验证,与蛋白质组结果一致,证实了蛋白质组技术的可靠性；(2)这些差异表达蛋白质可能是PPFP基因调控的蛋白质,与PPFP基因促进细胞增殖能力及迁徙运动能力密切相关,为筛选FTC的标志性蛋白或新的药物靶标蛋白提供了线索,为进一步阐明滤泡状甲状腺癌发生发展的分子机制奠定了基础。更多还原

【Abstract】 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.更多还原