本文選題：PPFP + 濾泡狀甲狀腺癌　； 參考：《中南大學》2010年博士論文

【摘要】： 研究背景 甲狀腺濾泡狀癌(follicular thyroid carcinoma,FTC)是最常見的甲狀腺惡性腫瘤之一,約占分化型甲狀腺癌的10%-30%,但其死亡率卻占甲狀腺癌總死亡率的40%以上。細針穿刺細胞學檢查及術中快速冰凍切片是鑒別甲狀腺結節(jié)良惡性的最有效方法,然而對于濾泡狀癌和甲狀腺腺瘤的鑒別卻仍存在一定的困難。FTC易發(fā)生血行轉移,早期即可通過血行播散轉移至肺、骨骼和腦等部位；未轉移時只有在手術切除的標本上發(fā)現(xiàn)包膜和血管侵犯方能確診,因此誤診率高,預后并不樂觀,嚴重危害人民群眾健康。因此探討FTC發(fā)生發(fā)展的分子機制,篩選FTC相關的標志蛋白,對于FTC的早期診治和改善預后均有重要意義。 甲狀腺特異轉錄因子PAX8與過氧化物酶增殖激活受體γ(PPARy)的融合基因,即PAX8/PPARγ融合基因(PPFP基因)是近年來發(fā)現(xiàn)的與FTC發(fā)生、發(fā)展密切相關的致瘤基因,已有不少研究證實,PPFP在濾泡狀甲狀腺癌中的陽性表達率明顯高于濾泡狀腺瘤,而在乳頭狀癌、嗜酸細胞腫瘤、未分化癌中未見PPFP的表達,因此認為PPFP融合基因的出現(xiàn)高度預示甲狀腺惡性病變,該融合基因可能會對甲狀腺癌的鑒別診斷和治療有幫助,并正逐漸成為研究FTC發(fā)病機制新的切入口和聚焦點。但PPFP基因在FTC發(fā)生發(fā)展中的具體作用及其機制尚不明了,值得進一步研究。 目的：構建含PPFP基因的慢病毒表達載體,并建立PPFP基因穩(wěn)定表達細胞株,為進一步探討PPFP基因?qū)θ苏＜谞钕偌毎飳W特性的影響及其機制奠定基礎。 方法：從含有PAX8基因和PPARy基因的質(zhì)�？寺∧０錚AX8-pOTB7和PPARγ-pCMV-SPORT6中,利用PCR方法分別釣取目的基因PAX8和PPARγ,將PAX8和PPARγ定向連接后克隆到pEGFP-C1載體上,構建PAX8和PPARy融合基因的真核表達質(zhì)粒pEGFP-C1-PPFP,并轉化大腸桿菌E. coli DH5 a,再通過PCR、測序和分析比對驗證PPFP基因。然后從已構建好的含PPFP基因的質(zhì)�？寺∧０錚EGFP-C1-PPFP中,利用PCR方法調(diào)取目的基因PPFP,將該基因克隆到慢病毒載體表達質(zhì)粒pGC-FU(含F(xiàn)lag基因)中,得到重組的pGC-FU-PPFP,通過PCR、測序和分析比對驗證PPFP基因后,將pGC-FU-PPFP質(zhì)粒和包裝質(zhì)粒pHelper1.0、pHelper2.0共同轉染人胚胎腎上皮細胞株293T細胞,獲得攜帶PPFP基因的重組慢病毒,收集并濃縮病毒上清液,測定重組慢病毒的滴度。再以攜帶PPFP基因的重組慢病毒感染目的細胞SV40大T抗原永生化人正常甲狀腺細胞系Nthy-ori 3-1,觀察感染效率。選擇感染效率滿意的細胞通過RT-PCR和Western blot多次檢測PPFP的表達,以確定PPFP基因穩(wěn)定表達細胞株的建立。 結果：(1)PCR方法成功獲得目的基因片段PAX8-1-7, PAX8-9及PPARy-1-6,并成功將PAX8和PPARy拼接,構建了PAX8與PPARy的融合基因的重組真核表達質(zhì)粒pEGFP-C1-PAX8/PPARγ。(2)以重組真核表達質(zhì)粒pEGFP-C1-PAX8/PPARγ為模板,PCR方法成功獲得目的基因PPFP,并成功構建PPFP基因的重組慢病毒表達質(zhì)粒pGC-FU-PPFP,以該質(zhì)粒與包裝質(zhì)粒共轉染293T細胞,成功獲得攜帶PPFP基因的高滴度的重組慢病毒,病毒滴度達3.5×107TU/mL。(3)以攜帶PPFP基因的重組慢病毒和空白慢病毒分別感染人正常甲狀腺細胞Nthy-ori 3-1,感染效率高,大于90%,以RT-PCR和Western blot檢測證實PPFP基因在mRNA和蛋白水平順利表達,PPFP基因穩(wěn)定表達細胞株成功建立,為進一步研究PPFP基因的功能及其作用機制奠定了基礎。 結論：(1)成功克隆了人PAX8與PPARy的融合基因(PPFP基因),并構建了PPFP基因的重組慢病毒載體；(2)慢病毒載體是理想的基因轉移載體,可以高效的將PPFP基因轉染至人正常甲狀腺細胞Nthy-ori 3-1,轉染后PPFP基因可持續(xù)穩(wěn)定表達。 目的：探討PPFP基因轉染對人正常甲狀腺細胞Nthy-ori 3-1生物學特性的影響,以明確PPFP基因在FTC發(fā)生發(fā)展過程中的作用。 方法：以PPFP基因穩(wěn)定表達細胞株Nthy-ori3-1PPFP、空白慢病毒感染細胞株Nthy-ori3-1Vector以及未感染細胞株Nthy-ori 3-1為研究對象,通過MTT檢測各組細胞增殖能力、平板克隆形成實驗檢測各組細胞體外克隆形成能力、軟瓊脂集落形成實驗檢測各組細胞停泊非依賴性生長、劃痕愈合實驗觀察各組細胞體外遷徙運動能力以及流式細胞儀檢測細胞凋亡和細胞周期,觀察PPFP基因轉染前后Nthy-ori 3-1細胞生物學特性的改變。 結果：(1)PPFP基因轉染可促進人甲狀腺細胞Nthy-ori 3-1增殖。(2)PPFP基因轉染可提高Nthy-ori 3-1細胞體外克隆形成能力。(3)PPFP基因轉染可促進Nthy-ori 3-1細胞的停泊非依賴性生長。(4)PPFP基因轉染可增強Nthy-ori 3-1細胞體外遷徙運動能力。(5)PPFP基因轉染可使Nthy-ori 3-1細胞周期進程加快,細胞G0/G1期細胞減少,S期及G2/M期細胞增加,并大大降低Nthy-ori 3-1細胞的凋亡率。 結論：PPFP基因轉染可顯著抑制人正常甲狀腺細胞Nthy-ori 3-1凋亡,并顯著促進人正常甲狀腺細胞Nthy-ori 3-1的增殖能力和遷徙運動能力,提示該基因可能在濾泡狀甲狀腺癌惡性轉化中起關鍵性作用。 目的：探討PPFP基因發(fā)揮致瘤作用的具體機制,篩選PPFP基因調(diào)控蛋白,以期為尋找FTC的標志性蛋白或新的藥物靶標蛋白提供線索。 方法：以蛋白質(zhì)組技術探討轉染PPFP基因前后Nthy-ori 3-1細胞蛋白質(zhì)組分的改變。(1)采用2-DE技術分別制備PPFP基因穩(wěn)定表達細胞株Nthy-ori3-1PPFP、空白慢病毒感染細胞株Nthy-ori3-1Vector以及未感染細胞株Nthy-ori 3-1總蛋白質(zhì)的雙向凝膠電泳圖譜。(2)應用PDQuest軟件對雙向電泳圖譜進行比較分析,尋找差異表達的蛋白質(zhì)點。選擇Nthy-ori3-1PPFP與另外兩組細胞差異均在兩倍以上的38個蛋白質(zhì)斑點作為候選蛋白質(zhì)點。(3)采用MALDI-TOF-MS質(zhì)譜分析技術對這些差異表達的蛋白質(zhì)進行鑒定。(4)選取部分差異蛋白質(zhì)點,以Western blot檢測各蛋白質(zhì)在三組細胞中的表達水平。 結果：(1)采用2-DE技術建立了Nthy-ori 3-1、Nthy-ori3-1Vector和Nthy-ori3-1PPFP三組細胞的雙向凝膠電泳圖譜。(2)應用PDQuest軟件分析發(fā)現(xiàn),每張2-DE圖譜均有數(shù)百個蛋白質(zhì)點,大多數(shù)蛋白質(zhì)點在位置和豐度上是一致的；Nthy-ori 3-1和Nthy-ori3-1Vector細胞2-DE圖譜的蛋白質(zhì)點無明顯差別,絕大部分點在位置和豐度上均一致；而Nthy-ori3-1PPFP與上述兩組細胞相比,存在一些差異較為明顯的點。(3) PDQuest軟件共發(fā)現(xiàn)Nthy-ori3-1PPFP與另外兩組細胞差異均在兩倍以上的38個蛋白質(zhì)斑點,采用MALDI-TOF-MS質(zhì)譜分析技術結合數(shù)據(jù)庫的搜索,共鑒定出了28個差異蛋白質(zhì)點。其中,Nthy-ori3-1PPFP與另外兩組相比,表達上調(diào)的點有19個,表達下調(diào)的點有9個。(4)選取其中MALDI-TOF-MS鑒定分數(shù)較高,覆蓋率較大,且認為與FTC發(fā)生發(fā)展關系較為密切的5個蛋白質(zhì)(Prohibitin、Galectin-1、Cytokeratin 8、Cytokeratin19、HSP27)作為驗證對象,Western blot方法檢測發(fā)現(xiàn),與Nthy-ori 3-1、Nthy-ori3-1Vector組相比,Prohibitin在Nthy-ori3-1PPFP組細胞中表達下調(diào),Galectin-1、Cytokeratin 8、Cytokeratin19、HSP27在Nthy-ori3-1PPFP組細胞中表達上調(diào),這與蛋白質(zhì)組學研究結果完全一致。 結論：(1)采用2-DE技術結合質(zhì)譜鑒定技術共篩選出PPFP基因轉染組細胞較對照組細胞28個差異表達蛋白質(zhì),并對部分差異蛋白質(zhì)的表達水平進行了驗證,與蛋白質(zhì)組結果一致,證實了蛋白質(zhì)組技術的可靠性；(2)這些差異表達蛋白質(zhì)可能是PPFP基因調(diào)控的蛋白質(zhì),與PPFP基因促進細胞增殖能力及遷徙運動能力密切相關,為篩選FTC的標志性蛋白或新的藥物靶標蛋白提供了線索,為進一步闡明濾泡狀甲狀腺癌發(fā)生發(fā)展的分子機制奠定了基礎。
[Abstract]:Background of the study



Thyroid carcinoma ( FTC ) is one of the most common thyroid malignant tumors , accounting for 10 - 30 % of the differentiated thyroid cancer , but its mortality is more than 40 % of the total mortality of thyroid cancer .
Therefore , it is very important to study the molecular mechanism of FTC ' s development and to screen the marker protein related to FTC . It is of great significance to the early diagnosis and treatment of FTC and to improve the prognosis .



PPFP fusion gene ( PPFP gene ) is the fusion gene which is closely related to the occurrence and development of FTC in thyroid carcinoma , and the expression of PPFP fusion gene is higher than that of follicular adenoma .



Objective : To construct a slow virus expression vector containing PPFP gene and establish a stable expression cell line of PPFP gene , which lays a foundation for further exploring the effect of PPFP gene on the biological characteristics of human normal thyroid cells and its mechanism .



Methods : The pGC - FU - PPFP was cloned into plasmid pGC - FU ( containing Flag gene ) of pGC - FU - PPFP and pGC - FU - PPFP was cloned into plasmid pGC - FU ( containing Flag gene ) of pGC - FU - PPFP .



Results : ( 1 ) The recombinant eukaryotic expression vector pGC - FU - PPFP of PPFP gene was successfully constructed by PCR method . The recombinant lentivirus with PPFP gene was successfully constructed by PCR and Western blot .



Conclusion : ( 1 ) The fusion gene ( PPFP gene ) of human PAX 8 and PPARy was cloned successfully , and the recombinant lentivirus vector of PPFP gene was constructed .
( 2 ) The slow virus vector is the ideal gene transfer vector , and the PPFP gene can be efficiently transfected into human normal thyroid cells Nthy - ori 3 - 1 , and the PPFP gene can be stably expressed after transfection .



Objective : To investigate the effect of PPFP gene transfection on the biological characteristics of human normal thyroid cells Nthy - ori 3 - 1 , and to clarify the role of PPFP gene in the development of FTC .



Methods : Nthy - ori3 - 1PPFP , Nthy - ori3 - 1Vector and Nthy - ori 3 - 1 , which were stably expressed by PPFP gene , were used as the research subjects . The proliferation ability of each group was detected by MTT assay . The ability of cell proliferation and cell cycle of each group were detected by MTT assay , and the changes of biological characteristics of Nthy - ori 3 - 1 cells were observed before and after transfection of PPFP gene .



Results : ( 1 ) PPFP gene transfection can promote the proliferation of human thyroid cells Nthy - ori 3 - 1 . ( 2 ) The transfection of PPFP gene can promote the growth of Nthy - ori 3 - 1 cell in vitro .



Conclusion : PPFP gene transfection can significantly inhibit the apoptosis of human normal thyroid cells Nthy - ori 3 - 1 and significantly promote the proliferation and migration ability of Nthy - ori 3 - 1 in normal thyroid cells , suggesting that the gene may play a key role in malignant transformation of follicular thyroid cancer .



Objective : To investigate the specific mechanism of PPFP gene to play the role of oncogenic tumor , and to screen PPFP gene regulatory protein , in order to provide clues for finding the marker protein of FTC or new drug target protein .



Methods : The protein components of Nthy - ori3 - 1PPFP , Nthy - ori3 - 1Vector and Nthy - ori 3 - 1 were prepared by using the technique of 2 - DE .



Results : ( 1 ) Two - dimensional gel electrophoresis of Nthy - ori 3 - 1 , Nthy - ori3 - 1Vector and Nthy - ori3 - 1PPFP was established by 2 - DE technique .
There was no significant difference between Nthy - ori 3 - 1 and Nthy - ori3 - 1 Vector cell 2 - DE . Most of them were consistent in position and abundance ;
The results showed that Nthy - ori3 - 1PPFP and Nthy - ori3 - 1PPFP showed down - regulation in Nthy - ori3 - 1PPFP group , and the expression of Galectin - 1 , Cytokeratin 8 , Cytokeratin 19 and HSP27 was up - regulated in Nthy - ori3 - 1PPFP group .



Conclusion : ( 1 ) There were 28 differentially expressed proteins in the transfected group of PPFP gene by using 2 - DE technique combined with mass spectrometry , and the expression level of partially differentiated protein was verified . The results were consistent with the results of protein group , which confirmed the reliability of protein group technology .
( 2 ) These differentially expressed proteins may be a protein regulated by PPFP gene , which is closely related to PPFP gene to promote cell proliferation and migration ability , provides clues for screening the marker protein of FTC or new drug target protein , and lays a foundation for further clarifying the molecular mechanism of development of follicular thyroid cancer .
【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R346

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本文編號：1972940