【摘要】：該博士論文由相互聯(lián)系的三部分組成。第一部分 遠處轉(zhuǎn)移性甲狀腺癌(DM-DTC)TERT及BRAF突變與碘難治關系研究端粒酶反轉(zhuǎn)錄酶(telomerase reverse transcriptase,TERT)啟動子突變在一些研究中報道與分化型甲狀腺癌(differentiated thyroid cancer,DTC)的侵襲性特征如復發(fā)、死亡等相關。然而其與遠處轉(zhuǎn)移性DTC(distant metastatic DTC,DM-DTC)遠處轉(zhuǎn)移病灶的攝1311特征及1311治療反應之間的關系尚未可知。本論文第一部分的研究目的為在中國DM-DTC患者人群中檢測TERT啟動子的突變率,并與BRAF突變相比較,評價其與遠處轉(zhuǎn)移病灶的攝131I特征及1311治療反應之間的關系。本研究回顧性收集了 66例DM-DTC患者的手術原發(fā)病灶石蠟切片組織,提取基因組DNA,采用PCR擴增及Sanger測序技術進行TERT啟動子及BRAF突變檢測,中位隨訪時間為46.5個月(四分位數(shù)間距:29個月～70.5個月)。在隨訪終點,根據(jù)隨訪過程中的血清學反應[刺激性甲狀腺球蛋白(stimulated thyroglobulin,sTg)水平的變化]、1311全身顯像所示攝碘特征(攝1311或不攝1311)及其他影像學證據(jù),將1311治療反應分為:①碘難治,②碘治療反應佳。對其中的36例患者的131I全身顯像攝碘情況進行了半定量分析,得到腫瘤病灶攝取量/本底攝取量(tumor/background,T/B)。本研究結(jié)果顯示:TERT啟動子突變在DM-DTC中的突變率為22.73%(15/66),其中C228T位點的突變率(13/15)比C250T位點(2/15)更高。TERT啟動子突變的患者有93.33%(14/15)在131I治療后出現(xiàn)sTg水平升高20%。而BRAF與TERT啟動子均不突變的患者有78.12%(25/32)在1311治療后出現(xiàn)sTg水平下降20%。TERT啟動子突變與DM-DTC患者1311治療反應差密切相關(P0.001),全部15例TERT啟動子突變的DM-DTC在隨訪終點均出現(xiàn)了碘難治,陽性預測值高達100%。TERT啟動子突變與DM-DTC的侵襲性特征如診斷時年齡大(P0.001)、腫瘤直徑大(P=0.013)、合并BRAF突變(P=0.044)顯著相關。對36例DM-DTC患者的1311全身顯像半定量分析提示:TERT啟動子突變與遠處轉(zhuǎn)移病灶的攝碘差顯著相關,其T/B水平明顯低于TERT野生組(P0.001)。另外,TERT啟動子突變的DM-DTC患者比BRAF突變的患者出現(xiàn)碘難治的時間更早(8/8vs.5/11,Fisher exact檢驗,P=0.018)。據(jù)此得出結(jié)論:TERT啟動子突變與DM-DTC遠處轉(zhuǎn)移病灶的不攝碘特征密切相關;且與BRAF相比,其對1311攝取特征的負性影響更加嚴重。TERT啟動子突變可作為識別碘難治性甲狀腺癌(radioiodine-refractory DTC,RAIR-DTC)的指標之一。第二部分miR-125a-3p與碘難治性甲狀腺癌(RAIR-DTC)關系研究目前國內(nèi)外關于DTC病灶1311攝取的相關小RNA(microRNA,miRNA)研究尚局限于體外細胞水平,缺乏RAIR-DTC腫瘤組織的miRNA表達譜研究。本論文第二部分研究的主要目的在于尋找DM-DTC遠處轉(zhuǎn)移病灶不同1311治療反應的患者,其手術原發(fā)病灶組織的miRNA表達譜的差異,以期為RAIR-DTC的識別提供分子依據(jù)。本研究回顧性篩選了接受至少兩次131I治療的DM-DTC患者99例。初始治療(甲狀腺全切術后+1311治療)后的中位隨訪時間為51.2個月。在隨訪終點,將所有患者根據(jù)1311治療影像學及血清學反應分為兩組:①碘難治,②碘治療反應佳。收集這些患者手術原發(fā)病灶石蠟包埋組織,提取總RNA。對質(zhì)量鑒定合格的88例進行后續(xù)實驗,其中56例進行miRNA表達譜芯片雜交,另外32例作為獨立樣本對芯片篩選的2個可能靶向調(diào)控鈉碘同向轉(zhuǎn)運體(sodium iodide symporter,NIS)mRNA表達的miRNA進行實時定量聚合酶鏈反應(quantitative real-time polymerase chain reaction,qRT-PCR)驗證,最終驗證得到1個在RAIR-DTC中高表達且可能靶向調(diào)控 NIS mRNA 的 miRNA:hsa-miR-125a-3p。在DTC細胞株B-CPAP中轉(zhuǎn)染miR-125a-3p的mimic、inhibitor及其各自陰性對照,結(jié)果顯示:轉(zhuǎn)染miR-125a-3p mimic較其陰性對照可顯著下調(diào)NIS mRNA及蛋白的表達水平;可顯著下調(diào)促甲狀腺激素受體(thyroid stimulating hormone receptor,TSHR)mRNA的表達水平,但對TSHR蛋白的影響不顯著;對甲狀腺球蛋白(thyroglobulin,TG)及甲狀腺過氧化物酶(thyroidperoxidase,TPO)mRNA及蛋白的影響不顯著;B-CPAP細胞的1311攝取率較其陰性對照顯著下降。轉(zhuǎn)染miR-125a-3p inhibitor較其陰性對照可顯著上調(diào)NIS mRNA及蛋白的表達水平;可顯著上調(diào)TSHR mRNA的表達水平,但對TSHR蛋白的影響不顯著;對TG及TPO mRNA及蛋白的影響不顯著;B-CPAP細胞的1311攝取率較其陰性對照顯著升高。據(jù)此得出結(jié)論:hsa-miR-125a-3p可通過抑制NIS的表達導致DTC細胞的131I攝取率下降,與RAIR-DTC的發(fā)生相關。第三部分1311治療前刺激性甲狀腺球蛋白可預測分化型甲狀腺癌治療反應研究提示sTg在1311治療后可有效監(jiān)測DTC的復發(fā)及疾病持續(xù)狀態(tài),但其在1311治療前評估中的作用尚未可知。有研究顯示1311治療前刺激性甲狀腺球蛋白(preablative sTg,ps-Tg)水平與DTC的遠處轉(zhuǎn)移相關,然而ps-Tg與131I治療反應的關系尚未可知。本論文第三部分的目的為研究ps-Tg與2015年美國甲狀腺協(xié)會(AmericanThyroidAssociation,ATA)指南提出的131I治療反應之間的關系,并計算可預測1311治療影像學反應不佳的ps-Tg臨界值。本研究回顧性研究了在北京協(xié)和醫(yī)院核醫(yī)學科甲狀腺癌全切術后接受131I治療的患者。除去甲狀腺球蛋白抗體(anti-Tgantibody,TgAb)陽性的患者,將452例DTC 患者按照 ps-Tg 水平分為 3 組:1 組,0～1 ng/ml(n=82);2 組,1～10ng/ml(n=173);3組,≥10ng/ml(n=197)。中位隨訪38個月后,按照2015年ATA指南治療反應分級,將治療反應分為療效滿意(excellentresponse,ER)、療效不確切(indeterminate response,IDR)、血清學反應欠佳(biochemical incomplete response,BIR)和影像學反應欠佳(strctural incomplete response,SIR)。其中,SIR 為反應差。Ps-Tg水平可明顯區(qū)分不同的治療反應。在隨訪終點,1組、2組、3組分別有0%、1.73%、42.74%患者的治療反應為SIR(x2=123.037,P0.001)。通過受試者工作特征曲線(receiver operating characteristic,ROC)計算得到區(qū)分SIR與其他治療反應(ER+IDR+BIR)的 ps-Tg 臨界值為 26.75ng/ml,曲線下面積(area under curve,AUC)為 0.947,陰性預測值(negative predictive value,NPV)為 96.99%。Ps-Tg為預測SIR的獨立預測因子(OR:42.312,P0.001)。Ps-Tg,尤其是ps-Tg高水平,可有效預測不同的1311治療反應,為1311治療決策的制定提供增益價值。
[Abstract]:The doctoral thesis consists of three interrelated parts. Part 1 the relationship between TERT and BRAF mutations and iodine refractory mutations in distant metastatic thyroid carcinoma (DM-DTC) study of the promoter mutation of the telomerase reverse transcriptase (telomerase reverse transcriptase, TERT) promoter in some studies and differentiated thyroid cancer (differentiated thyroid cancer, DTC) Invasive characteristics such as recurrence and death are related. However, the relationship between the 1311 features of distant metastatic DTC (distant metastatic DTC, DM-DTC) and the 1311 treatment response is not known. The first part of this thesis is to detect the mutation rate of the TERT promoter in the Chinese population of DM-DTC patients and to be with BRAF process. A retrospective study was made to evaluate the relationship between the 131I feature and the 1311 treatment response. In this study, the paraffin section of the primary lesions of 66 patients with DM-DTC was collected, the genomic DNA was extracted and the TERT promoter and BRAF mutation were detected by PCR amplification and Sanger sequencing. The median follow-up time was 46.5. A month (four division interval: 29 months to 70.5 months). At the end of the follow-up, according to the serological response [stimulating thyroid globulin (stimulated thyroglobulin, sTg) levels in the follow-up process], 1311 whole body imaging showed the iodine characteristics (1311 or no 1311) and other imaging evidence, and the 1311 treatment reactions were divided into: 1. Iodine refractory, A semi quantitative analysis of iodine uptake in 36 cases of 131I was carried out and the uptake of tumor lesions / tumor/background (T/B) was obtained. The results of this study showed that the mutation rate of the TERT promoter mutation in DM-DTC was 22.73% (15/66), and the mutation rate of the C228T locus (13/15) was more than the C250T site. (2/15) 93.33% (14/15) in patients with higher.TERT promoter mutation (14/15) increased sTg level after 131I and 78.12% (25/32) in patients with BRAF and TERT promoter without mutation (25/32), the decrease of sTg level after 1311 treatment was closely related to the poor response of 1311 of DM-DTC patients. The mutation of DM-DTC appeared at the end point of the follow-up. The positive predictive value was up to the 100%.TERT promoter mutation and the invasive characteristics of DM-DTC, such as the age of diagnosis (P0.001), the large diameter of the tumor (P=0.013), and the combined BRAF mutation (P=0.044). A semi quantitative analysis of the 1311 body imaging of 36 cases of DM-DTC patients: TERT promoter mutation The T/B level was significantly lower than that in the TERT wild group (P0.001). In addition, the DM-DTC patients with TERT promoter mutation appeared earlier than those with BRAF mutations (8/8vs.5/11, Fisher exact test, P=0.018). Accordingly, the conclusion was that the TERT promoter mutation and the distant metastasis of the DM-DTC were not taken. Iodine characteristics are closely related; and compared with BRAF, the negative effect on the 1311 uptake characteristics is more serious..TERT promoter mutation can be used as one of the indicators for identifying iodine refractory thyroid carcinoma (radioiodine-refractory DTC, RAIR-DTC). The relationship between the second part of miR-125a-3p and iodized thyroid adenocarcinoma (RAIR-DTC) is a study of DTC disease at home and abroad. The study of small RNA (microRNA, miRNA) uptake of focal 1311 is still limited to the level of in vitro cells and the lack of miRNA expression profiles in RAIR-DTC tumor tissues. The main purpose of the second part of this study is to find the differences in the miRNA expression profiles of the primary foci in the patients with distant metastatic lesions of DM-DTC. This study provides a molecular basis for RAIR-DTC identification. This study reviewed 99 cases of DM-DTC patients receiving at least two 131I treatments. The median follow-up time after initial treatment (+1311 after total thyroidectomy) was 51.2 months. All patients were divided into two groups according to the 1311 treatment imaging and serological response at the end of the follow-up: 1. Treatment, iodine therapy was better. Collect the paraffin embedded tissues of the primary foci of these patients, and extract the total RNA. for 88 cases of quality identification. 56 of them were hybridized with miRNA expression spectrum chip, and the other 32 were selected as independent samples to regulate the sodium iodide symporte (iodide symporte). R, NIS) mRNA expressed miRNA was verified by real-time quantitative polymerase chain reaction (quantitative real-time polymerase chain reaction, qRT-PCR). Negative control, the results showed that transfection of miR-125a-3p mimic significantly down the expression level of NIS mRNA and protein compared with the negative control, and significantly down regulation of the expression level of thyroid stimulating hormone receptor (thyroid stimulating hormone receptor, TSHR) mRNA, but no significant effect on TSHR protein; The effect of thyroidperoxidase (TPO) mRNA and protein was not significant. The 1311 uptake rate of B-CPAP cells was significantly lower than that of the negative control. The expression level of NIS mRNA and protein could be significantly up-regulated by transfection of miR-125a-3p inhibitor compared with the negative control, and the expression level of TSHR mRNA was significantly up, but the effect on TSHR protein was not obvious. The effect on TG and TPO mRNA and protein was not significant; the 1311 uptake rate of B-CPAP cells was significantly higher than that of the negative control. Accordingly, it was concluded that hsa-miR-125a-3p could decrease the 131I uptake rate of DTC cells by inhibiting the expression of NIS and related to the occurrence of RAIR-DTC. Third part of the stimulative thyroglobulin can be predicted before the treatment of part 1311. The study of therapeutic response to thyroid cancer suggests that sTg can effectively monitor the recurrence and disease status of DTC after 1311 treatment, but its role in the assessment before 1311 is not known. Studies have shown that the level of preablative sTg (ps-Tg) before 1311 treatment is related to the distant metastasis of DTC, but ps-Tg and 131I therapy The third part of this paper aims to study the relationship between ps-Tg and the AmericanThyroidAssociation (ATA) guidelines of the American Thyroid Association (AmericanThyroidAssociation, ATA) in 2015, and to calculate the critical value of ps-Tg for predicting the poor response of the 1311 treatment imaging. This study reviewed the Peking Union Medical College Hospital in a retrospective study. The patients who received 131I after total thyroidectomy in the nuclear medicine department were divided into 3 groups according to ps-Tg level, excluding the positive of thyroid globulin antibody (anti-Tgantibody, TgAb). The 1 group, 0~1 ng/ml (n=82), the 2 group, 1 to 10ng/ml (n=173), 3 groups, and more 10ng/ml (n=197) were followed up for 38 months after the median follow-up, according to the ATA guide of 2015. The treatment reaction classification was divided into excellentresponse (ER), the curative effect was not accurate (indeterminate response, IDR), the serological response was not good (biochemical incomplete response, BIR) and the imaging reaction was not good (strctural incomplete response). The therapeutic response was SIR (x2=123.037, P0.001) in 0%, 1.73%, and 42.74% patients in the 1 and 2 groups at the end point of follow-up. The ps-Tg critical value of the SIR and other therapeutic reactions (ER+ IDR+BIR) was calculated by the work characteristic curve of the subjects (receiver operating characteristic, ROC). Urve, AUC) is 0.947, and the negative predictive value (negative predictive value, NPV) is 96.99%.Ps-Tg as the independent predictor of SIR (OR:42.312, P0.001).Ps-Tg, especially the ps-Tg high level, which can effectively predict different 1311 treatment responses and provide gain value for the formulation of 1311 treatment decisions.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R736.1

【相似文獻】

相關期刊論文 前10條

1 Ting Tan;Zhi-Qi Hu;;Construction and expression of retroviral vector pLEGFP-N1-TERT in preparation of seed cells for skin tissue engineering[J];Asian Pacific Journal of Tropical Medicine;2013年12期

2 陳華潔;余日安;賀凌飛;夏濤;吳志剛;楊克敵;陳學敏;;硒和鎘聯(lián)合作用對大鼠肝臟TERT mRNA表達的影響[J];毒理學雜志;2006年02期

3 戴文濤;陳華潔;余日安;賀凌飛;陳秉;陳學敏;;Effects of Cadmium on Telomerase Activity,Expressions of TERT,C-myc and P53,and Apoptosis of Rat Hepatocytes[J];Journal of Huazhong University of Science and Technology(Medical Sciences);2010年06期

4 杜鵑;馬玉實;范志朋;;TERT過表達對人骨髓間充質(zhì)干細胞精氨酸組蛋白甲基化酶表達影響的研究[J];北京口腔醫(yī)學;2012年04期

5 黃煒;呂安林;劉博武;侯靜;侯兆蕾;達晶;侯紅;;Plenti6.3/V5-TERT慢病毒載體的構建并成功建立永生化大鼠骨髓間充質(zhì)干細胞系的研究[J];中華臨床醫(yī)師雜志(電子版);2012年10期

6 曹陽,黃巨恩;TERT—原位雜交技術的使用難點及解決方法[J];廣西醫(yī)科大學學報;2004年05期

7 吳麗娜;魏林;楊建嶺;石文凱;霍明山;李彩霞;崔玉秀;曾瑞紅;;TERT多表位疫苗的制備及其免疫原性初步研究[J];中國免疫學雜志;2010年10期

8 張光謀;吳俊琢;徐振平;郭志坤;王鵬;;胎兒心肌細胞TERT和cyclinD1的表達[J];解剖學研究;2005年04期

9 程文;位志峰;高建平;張征宇;葛京平;景抗震;徐鋒;解鵬;;Effects of Combined siRNA-TR and-TERT on Telomerase Activity and Growth of Bladder Transitional Cell Cancer BIU-87 Cells[J];Journal of Huazhong University of Science and Technology(Medical Sciences);2010年03期

10 梁曉慧;韓璐;李素華;徐新娟;;TERT基因單核苷酸多態(tài)性與新疆維吾爾族人長壽的關聯(lián)研究[J];新疆醫(yī)科大學學報;2011年10期

相關會議論文 前7條

1 曾瑞紅;吳麗娜;楊建嶺;魏林;李彩霞;崔玉秀;;TERT多表位疫苗的制備及其免疫原性初步研究[A];河北省免疫學會第六次免疫學大會資料匯編[C];2010年

2 王琰;徐瑞榮;周延峰;劉朝霞;;端粒酶逆轉(zhuǎn)錄酶TERT在獲得性再生障礙性貧血患者中的表達研究[A];中華中醫(yī)藥學會第二屆岐黃論壇——血液病中醫(yī)藥防治分論壇論文集[C];2014年

3 陳晨;張逸杰;徐勇;王燕;黃世勇;陳始明;肖伯奎;陶澤璋;;c-fos和c-jun二聯(lián)體在TERT促進多種腫瘤細胞增殖過程中的作用研究[A];2010全國耳鼻咽喉頭頸外科中青年學術會議論文匯編[C];2010年

4 戴文濤;賀凌飛;余日安;陳秉;;鎘對大鼠肝細胞端粒酶活性、凋亡和TERT、c-Myc和p53表達的影響[A];廣東省環(huán)境誘變劑學會、廣東省預防醫(yī)學會衛(wèi)生毒理專業(yè)委員會2010年學術會議資料匯編[C];2010年

5 趙宏賢;王巧稚;徐富翠;余鴻;;DNA去甲基化對大鼠骨髓間充質(zhì)干細胞TERT的影響[A];中國解剖學會2011年年會論文文摘匯編[C];2011年

6 鄭玲玲;蔡琳;;5p15.33(TERT-CLPTM1L)、15q25.1(CHRAN3)基因多態(tài)性與非吸煙者肺癌的關聯(lián)研究[A];全國腫瘤流行病學和腫瘤病因?qū)W學術會議論文集[C];2011年

7 李嬌;屈藝;母得志;;大鼠端粒酶逆轉(zhuǎn)錄酶對神經(jīng)元缺氧缺血性損傷的保護作用[A];中華醫(yī)學會第十七次全國兒科學術大會論文匯編（上冊）[C];2012年

相關博士學位論文 前10條

1 宋勇莉;TERT促耳蝸前體細胞增殖及其機制研究[D];第四軍醫(yī)大學;2015年

2 譚卿;尿路上皮腫瘤TERT啟動子突變的研究[D];北京協(xié)和醫(yī)學院;2016年

3 江洋;端粒酶逆轉(zhuǎn)錄酶TERT促進人喉癌細胞Hep-2增殖作用及機制的研究[D];武漢大學;2013年

4 李瑞;TERT啟動子突變在膠質(zhì)瘤中的分布以及預后分層研究[D];南京醫(yī)科大學;2016年

5 李小廷;候選SNPs與肺癌的關聯(lián)分析及作用機制的研究[D];天津醫(yī)科大學;2015年

6 溫強;BRAF V600E和TERT啟動子突變在PTC術后患者核素治療中的作用研究[D];吉林大學;2017年

7 楊雪;分子特征在識別碘難治性甲狀腺癌中的意義[D];北京協(xié)和醫(yī)學院;2017年

8 李曉燕;TERT基因轉(zhuǎn)染骨髓內(nèi)皮祖細胞移植對5/6腎切除大鼠腎臟損傷的修復研究[D];中南大學;2012年

9 吳小琴;端粒酶逆轉(zhuǎn)錄酶TERT通過與NF-kappa B通路的正反饋調(diào)控酒精性肝病中巨噬細胞極化的作用機制研究[D];安徽醫(yī)科大學;2017年

10 楊平勛;TERT在腫瘤細胞中保護染色體末端的功能機制研究[D];中國人民解放軍軍事醫(yī)學科學院;2011年

相關碩士學位論文 前10條

1 何潔;膠質(zhì)瘤中TERT啟動子區(qū)突變對預后的影響[D];安徽醫(yī)科大學;2015年

2 易忠良;環(huán)黃芪醇對腦缺血再灌注大鼠神經(jīng)元凋亡和TERT表達的影響[D];承德醫(yī)學院;2015年

3 陳金生;膠質(zhì)瘤干細胞誘導骨髓間充質(zhì)干細胞惡性轉(zhuǎn)化的實驗研究[D];蘇州大學;2016年

4 盧保德;廣西地區(qū)人群XRCC4、TERT基因單核苷酸多態(tài)性與膀胱癌易感性的關系[D];廣西醫(yī)科大學;2016年

5 周子敬;宮頸病變及宮頸癌C13orf18、TERT及SLIT1基因甲基化水平及臨床意義[D];天津醫(yī)科大學;2016年

6 林孔英;HBV DNA在端粒酶逆轉(zhuǎn)錄酶基因組上整合及其整合后表達調(diào)控機制[D];福建醫(yī)科大學;2016年

7 張佩;TERT端粒酶非依賴機制與丙烯酰胺誘導的神經(jīng)元損傷[D];武漢科技大學;2014年

8 尋曉潔;TERT基因甲基化、突變及表達在腦膠質(zhì)瘤患者中的研究[D];西北大學;2015年

9 宋揚;RNA干擾TERT基因促進脊髓修復的體外實驗研究[D];新疆醫(yī)科大學;2014年

10 宋景春;TERT在5-HT誘導的肺動脈平滑肌細胞增殖過程中的作用及機制研究[D];第四軍醫(yī)大學;2004年

，

本文編號：2159451