發(fā)布時(shí)間：2018-04-29 11:58

  本文選題：谷胱甘肽過(guò)氧化物酶 + 7��； 參考：《大連醫(yī)科大學(xué)》2017年博士論文

【摘要】：研究背景:甲狀腺癌是內(nèi)分泌器官中最常見(jiàn)的惡性腫瘤,主要病理類型有甲狀腺乳頭狀癌(papillary thyroid carcinoma,PTC)、濾泡癌、未分化癌和髓樣癌等。其中PTC最為常見(jiàn)。一般而言,PTC發(fā)展緩慢,病程較長(zhǎng),預(yù)后較好,但也有很多PTC患者因沒(méi)有臨床癥狀,就診較晚,一經(jīng)發(fā)現(xiàn),即伴隨有淋巴結(jié)轉(zhuǎn)移、被膜侵犯等情況,導(dǎo)致患者的預(yù)后不佳;故仍需要早發(fā)現(xiàn)、早診斷、早治療PTC。盡管多數(shù)文獻(xiàn)報(bào)道,PTC的發(fā)病機(jī)制與BRAF基因突變、RET/PTC基因重排等有關(guān),但目前有關(guān)PTC的發(fā)病機(jī)制仍未完全明確,因此深入研究PTC的發(fā)病機(jī)制有著重要的臨床意義。哺乳動(dòng)物細(xì)胞中的谷胱甘肽過(guò)氧化物酶(glutathione peroxidase,GPXs)是機(jī)體內(nèi)最主要的氧化還原酶,由8種GPX組成,分別命名為GPX1-GPX8,其中GPX1-GPX4以及GPX6為含硒代半胱氨酸的GPXs,GPX5、GPX7及GPX8則為非含硒代半胱氨酸的GPXs。通常,含硒代半胱氨酸的GPXs能夠通過(guò)催化還原型谷胱甘肽(GSH)來(lái)還原H2O2,以達(dá)到降低疾病氧化應(yīng)激反應(yīng)的作用。而非含硒代半胱氨酸的GPXs,不同于含硒代半胱氨酸的GPXs,其缺少GSH結(jié)合結(jié)構(gòu)域,因此,它們引起過(guò)氧化物酶活性的機(jī)制還存在爭(zhēng)議�；钚匝醮�(reactive oxygen species,ROS)包括氧離子和過(guò)氧化物等活性含氧分子,是機(jī)體正常代謝產(chǎn)生的內(nèi)在產(chǎn)物。有學(xué)者提出氧化應(yīng)激反應(yīng)與腫瘤的發(fā)生、發(fā)展有關(guān)。所謂的氧化應(yīng)激反應(yīng)是指機(jī)體遭受有害刺激時(shí),體內(nèi)的ROS產(chǎn)生過(guò)多,使機(jī)體氧化程度超出氧化物的清除,使得氧化系統(tǒng)與抗氧化系統(tǒng)失衡而引起的組織損傷。ROS包括O2-、H2O2、HO2-、-OH等的活性含氧分子。長(zhǎng)期持續(xù)的氧化應(yīng)激,會(huì)導(dǎo)致自身免疫異常、腫瘤發(fā)生、糖尿病、肥胖、神經(jīng)變性和衰老等等多種疾病。甲狀腺的主要生理功能是合成甲狀腺激素,其中的h2o2作為甲狀腺激素合成的關(guān)鍵酶-甲狀腺過(guò)氧化物酶的必需底物,對(duì)甲狀腺激素合成及發(fā)揮正常甲狀腺生理功能起到非常重要的作用。當(dāng)機(jī)體發(fā)生異常時(shí),h2o2可在甲狀腺中過(guò)度蓄積,使得甲狀腺持續(xù)暴露在高濃度的h2o2及其附帶效應(yīng)產(chǎn)生的ros環(huán)境中,即氧化應(yīng)激反應(yīng)中,使甲狀腺細(xì)胞受到氧化性dna損傷或基因低甲基化。已有研究證實(shí),大量的ros可促進(jìn)自身細(xì)胞的增殖、侵襲、轉(zhuǎn)移及血管生成,并能逃避凋亡的發(fā)生。因此,氧化還原反應(yīng)的異�？赡芘cptc的發(fā)生、發(fā)展有關(guān)。gpx7是gpxs家族成員之一,主要參與維持機(jī)體的氧化還原穩(wěn)態(tài)的作用。近年來(lái)研究發(fā)現(xiàn),gpx7異常表達(dá)與多種腫瘤(包括食管腺癌、乳腺癌、肝細(xì)胞癌等)的發(fā)生、發(fā)展相關(guān),但有關(guān)gpx7在甲狀腺癌中的研究尚未見(jiàn)報(bào)道。已知nf-κb是一種存在于真核細(xì)胞中控制dna轉(zhuǎn)錄的重要核轉(zhuǎn)錄因子,具有多向轉(zhuǎn)錄調(diào)節(jié)作用,已證實(shí)nf-κb與多種惡性腫瘤的發(fā)生、發(fā)展有關(guān)。有文獻(xiàn)報(bào)道gpx7在食管腺癌(eac)中的作用有可能是通過(guò)nf-κb信號(hào)通路來(lái)發(fā)揮作用的。cyclind1能夠正性調(diào)控細(xì)胞周期,是檢測(cè)細(xì)胞增殖活性的指標(biāo)之一,有報(bào)道cyclind1是nf-κbp65的重要下游靶基因。有關(guān)gpx7在甲狀腺乳頭狀癌中與nf-κb、cyclind1表達(dá)關(guān)系的研究,尚未見(jiàn)報(bào)道。因此,本研究目的是從組織和細(xì)胞水平分別研究gpx7在ptc中的表達(dá),以及與ptc臨床病理參數(shù)的關(guān)系,探討gpx7在ptc發(fā)生、發(fā)展過(guò)程中的作用及可能機(jī)制,為進(jìn)一步研究gpx7在甲狀腺乳頭狀癌中的作用機(jī)制奠定基礎(chǔ)。目的:1、從組織和細(xì)胞水平研究gpx7在ptc中的表達(dá)情況及對(duì)ptc細(xì)胞增殖和凋亡的影響,探討gpx7在ptc發(fā)生、發(fā)展中所起的作用。2、從組織和細(xì)胞水平分別研究gpx7與nf-κbp65、cyclind1表達(dá)的關(guān)系,探討gpx7參與調(diào)控ptc細(xì)胞增殖與凋亡的可能機(jī)制。3、研究gpx7蛋白在伴有橋本甲狀腺炎的甲狀腺乳頭狀癌(ptc+ht)組織中的表達(dá),探討橋本甲狀腺炎的炎性微環(huán)境對(duì)gpx7表達(dá)的影響,為進(jìn)一步研究gpx7在甲狀腺乳頭狀癌中的作用機(jī)制奠定基礎(chǔ)。方法:1、用免疫組織化學(xué)方法檢測(cè)30例ptc、14例結(jié)節(jié)性甲狀腺腫(結(jié)甲)組織中g(shù)px7蛋白的表達(dá)情況,并分析gpx7與臨床病理特征的關(guān)系。2、構(gòu)建gpx7基因干擾慢病毒載體,用westernblot方法外源驗(yàn)證靶點(diǎn)有效后,用構(gòu)建成功的gpx7基因干擾慢病毒載體感染甲狀腺乳頭狀癌(k1)細(xì)胞,使用qt-pcr的方法檢測(cè)基因敲減效果;并用多種細(xì)胞功能實(shí)驗(yàn)(celigo細(xì)胞計(jì)數(shù)、流式細(xì)胞儀檢測(cè)、caspase3/7檢測(cè)、mtt檢測(cè)、細(xì)胞克隆形成實(shí)驗(yàn))方法,觀察gpx7基因敲減與未基因敲減的k1細(xì)胞的細(xì)胞增殖、凋亡、克隆形成的差異。3、用免疫組織化學(xué)方法檢測(cè)30例ptc、14例結(jié)節(jié)性甲狀腺腫(結(jié)甲)組織中nf-κbp65、cyclind1蛋白的表達(dá)情況,分析各指標(biāo)與gpx7表達(dá)的關(guān)系。4、用gpx7基因干擾慢病毒載體感染k1細(xì)胞,并用westernblot方法檢測(cè)gpx7基因敲減前后nf-κbp65、cyclind1蛋白表達(dá)情況。5、用免疫組織化學(xué)方法檢測(cè)29例伴有橋本甲狀腺炎的甲狀腺乳頭狀癌(ptc+ht組)、31例甲狀腺乳頭狀癌(ptc組)、16例橋本甲狀腺炎(ht組)以及14例結(jié)節(jié)性甲狀腺腫(結(jié)甲組)組織的gpx7表達(dá)情況,結(jié)合臨床病理參數(shù),探討橋本甲狀腺炎的炎性微環(huán)境對(duì)gpx7表達(dá)的影響及在甲狀腺乳頭狀癌中所起的作用。6、免疫組化評(píng)判標(biāo)準(zhǔn):每例均隨機(jī)觀察5個(gè)高倍視野,根據(jù)染色細(xì)胞百分率和染色強(qiáng)度進(jìn)行評(píng)定和分析。陽(yáng)性細(xì)胞百分率計(jì)分標(biāo)準(zhǔn):10%為0分,11%~25%為1分,26%~50%為2分,51%~75%為3分,75%為4分;染色強(qiáng)度計(jì)分:無(wú)染色為0分,淺黃色為1分,棕黃色為2分,棕褐色為3分。將陽(yáng)性細(xì)胞率得分和染色強(qiáng)度得分相乘即為最后得分,0分為陰性(-),1~4分為弱陽(yáng)性(+),5~8分為中度陽(yáng)性(++),9~12分為強(qiáng)陽(yáng)性(+++)。7、統(tǒng)計(jì)學(xué)方法:采用spss20.0統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)分析,計(jì)量資料采用均數(shù)±標(biāo)準(zhǔn)差表示,組間差異比較采用獨(dú)立樣本t檢驗(yàn),如方差不齊則采用t’檢驗(yàn)。計(jì)數(shù)資料采用例(百分率)表示,組間比較采用卡方檢驗(yàn)。相關(guān)性分析采用pearson相關(guān)性分析。p0.05為差異具有統(tǒng)計(jì)學(xué)意義。結(jié)果:1、gpx7對(duì)甲狀腺乳頭狀癌細(xì)胞增殖、凋亡影響的研究(1)免疫組化結(jié)果:(1)gpx7表達(dá)于甲狀腺細(xì)胞胞質(zhì)中。gpx7在ptc組表達(dá)陽(yáng)性率為100.0%,以中-強(qiáng)陽(yáng)性為主;結(jié)甲組gpx7表達(dá)表達(dá)陽(yáng)性率為30.0%,以弱陽(yáng)性為主,ptc組gpx7表達(dá)明顯高于結(jié)甲組(p0.05)。(2)gpx7的表達(dá)與腫瘤最大徑呈正相關(guān)關(guān)系(r=0.601,p0.05),且gpx7高表達(dá)組腫瘤最大徑(1.56±0.56cm)明顯大于gpx7低表達(dá)組(0.56±0.13cm)(p0.05)。(2)細(xì)胞實(shí)驗(yàn)結(jié)果:(1)選取兩種人甲狀腺乳頭狀癌細(xì)胞系tpc-1和k1。qt-pcr結(jié)果顯示gpx7基因在tpc-1和k1細(xì)胞中都有較高的表達(dá)豐度。相比較k1細(xì)胞表達(dá)豐度更高,故選取k1細(xì)胞進(jìn)行后續(xù)實(shí)驗(yàn)研究。(2)gpx7基因干擾慢病毒載體制備后,采用westernblot外源驗(yàn)證,發(fā)現(xiàn)gpx7基因敲減后gpx7的表達(dá)明顯減少,說(shuō)明靶點(diǎn)對(duì)gpx7基因的外源表達(dá)有敲減作用,是有效靶點(diǎn)。(3)gpx7基因干擾慢病毒載體感染k1細(xì)胞后通過(guò)普通顯微鏡和熒光顯微鏡下觀察,發(fā)現(xiàn)細(xì)胞感染效率達(dá)到80%以上,且細(xì)胞狀態(tài)正常,采用qpcr的法證實(shí)經(jīng)shrna慢病毒感染后,k1細(xì)胞中g(shù)px7基因在mrna水平的表達(dá)量受到抑制,敲減效率達(dá)到81.5%,說(shuō)明載體感染k1細(xì)胞效果好,可以進(jìn)行后續(xù)的實(shí)驗(yàn)觀察。(4)celigo細(xì)胞計(jì)數(shù)結(jié)果顯示,gpx7基因敲減組細(xì)胞增殖數(shù)明顯減少,培養(yǎng)第5天較第1天僅增殖(5.00±0.32)倍,與對(duì)照組的(8.44±0.26)相比較,增殖速率明顯減慢。(5)流式細(xì)胞儀檢測(cè)發(fā)現(xiàn),gpx7基因敲減組細(xì)胞凋亡百分比(24.08±0.28)%,與對(duì)照組的(4.33±0.15)%相比較,凋亡比例明顯增多。(6)caspase3/7活性檢測(cè)發(fā)現(xiàn),gpx7基因敲減組的caspase3/7活性值為(257.82±21.28),與對(duì)照組的(100.00±5.38)相比較,明顯增加。(7)mtt檢測(cè)發(fā)現(xiàn),gpx7基因敲減組培養(yǎng)第5天od490值較第1天增長(zhǎng)(3.081±0.160)倍,與對(duì)照組(增長(zhǎng)4.897±0.091倍)相比較,gpx7基因敲減組細(xì)胞增長(zhǎng)倍數(shù)明顯減少。(8)細(xì)胞克隆形成檢測(cè)實(shí)驗(yàn)發(fā)現(xiàn),gpx7基因敲減組克隆形成數(shù)(283±6)與對(duì)照組克隆形成數(shù)(710±10)相比較,明顯減少。2、gpx7在甲狀腺乳頭狀癌中的可能作用機(jī)制(1)免疫組化結(jié)果(1)nf-κbp65表達(dá)于甲狀腺細(xì)胞胞質(zhì)和(或)細(xì)胞核中。ptc組表達(dá)明顯高于結(jié)甲組。ptc組nf-κbp65的陽(yáng)性表達(dá)率達(dá)83.3%,以中度陽(yáng)性為主。(2)cyclind1表達(dá)于甲狀腺細(xì)胞核。ptc組表達(dá)明顯高于結(jié)甲組。ptc組cyclind1陽(yáng)性表達(dá)率達(dá)到96.7%,以中度陽(yáng)性為主。(3)gpx7、nf-κbp65、cyclind1蛋白表達(dá)呈顯著正相關(guān)(r值分別為0.721、0.589、0.703,p值均0.05)。(4)ptc組gpx7與nf-κbp65聯(lián)合檢測(cè)陽(yáng)性率為83.3%,與cyclind1聯(lián)合檢測(cè)陽(yáng)性率為100.0%,三者聯(lián)合檢測(cè)陽(yáng)性率為83.3%。(2)細(xì)胞實(shí)驗(yàn)結(jié)果(1)慢病毒感染目的細(xì)胞效率較高,qpcr證實(shí)gpx7基因被有效敲減。(2)westernblot實(shí)驗(yàn)結(jié)果顯示,敲減甲狀腺乳頭狀癌細(xì)胞(k1細(xì)胞系)的gpx7基因后,nf-κbp65蛋白表達(dá)量無(wú)明顯變化,cyclind1蛋白表達(dá)量增高,與敲減前比較,差異有統(tǒng)計(jì)學(xué)意義(p0.05)。3、炎性微環(huán)境對(duì)gpx7在ptc中表達(dá)的影響結(jié)果(1)免疫組化結(jié)果顯示,伴橋本甲狀腺炎的甲狀腺乳頭狀癌組織gpx7蛋白表達(dá)評(píng)分(6.21±2.29)低于甲狀腺乳頭狀癌(8.52±2.41),差異有統(tǒng)計(jì)學(xué)意義(p0.05)。(2)單因素分析發(fā)現(xiàn)影響gpx7表達(dá)的因素有合并ht、tg-ab、tpo-ab、腫瘤部位和被膜侵犯情況,其中g(shù)px7與合并ht、tg-ab和tpo-ab水平呈負(fù)相關(guān),與腫瘤部位、被膜侵犯情況呈正相關(guān)。(3)多因素線性回歸分析發(fā)現(xiàn)影響gpx7表達(dá)的因素為有合并ht。(4)臨床病理特征分析,發(fā)現(xiàn)合并ht炎性環(huán)境的ptc腫瘤多局限于單側(cè),被膜侵犯發(fā)生率低。結(jié)論:1、免疫組化和細(xì)胞實(shí)驗(yàn)結(jié)果顯示GPX7在甲狀腺乳頭狀癌中高表達(dá)。2、免疫組化和細(xì)胞實(shí)驗(yàn)結(jié)果提示GPX7具有促進(jìn)甲狀腺乳頭狀癌細(xì)胞增殖、減少細(xì)胞凋亡的作用。3、GPX7、NF-κB p65、Cyclin D1蛋白在甲狀腺乳頭狀癌中的表達(dá)呈正相關(guān),提示三者在甲狀腺乳頭狀癌的發(fā)生、發(fā)展中可能有關(guān)聯(lián)。4、GPX7基因敲減后NF-κB p65蛋白表達(dá)無(wú)變化,提示GPX7對(duì)甲狀腺乳頭狀癌的作用與NF-κB信號(hào)通路無(wú)關(guān)或不是通過(guò)調(diào)節(jié)其蛋白表達(dá)量來(lái)實(shí)現(xiàn)的。5、GPX7基因敲減后Cyclin D1蛋白表達(dá)增高,機(jī)制不清。6、通過(guò)單因素和多因素線性回歸分析,發(fā)現(xiàn)是否合并橋本甲狀腺炎是影響甲狀腺乳頭狀癌組織中GPX7表達(dá)的因素,提示橋本甲狀腺炎的炎性環(huán)境能抑制甲狀腺乳頭狀癌組織中GPX7的表達(dá)。
[Abstract]:Background: thyroid cancer is the most common malignant tumor in the endocrine organs. The main pathological types are papillary thyroid carcinoma (PTC), follicular, undifferentiated and medullary carcinoma. Among them, PTC is the most common. Generally speaking, the development of PTC is slow, the course is longer, and the prognosis is better, but there are many PTC patients who have no presence. Bed symptoms, later diagnosis, once found, that is accompanied by lymph node metastasis, membrane invasion and other conditions, resulting in poor prognosis. Therefore, early detection, early diagnosis, early treatment of PTC., although most of the literature reports, the pathogenesis of PTC is related to the BRAF gene mutation, the RET/PTC gene rearrangement, but the pathogenesis of PTC is still not completely clear. It is true that it is important to study the pathogenesis of PTC. The glutathione peroxidase (GPXs) in mammalian cells is the most important oxidoreductase in the body, which is composed of 8 GPX, named GPX1-GPX8, and GPX1-GPX4 and GPX6 are GPXs, GPX5, GPX7, and selenocysteine containing selenocysteine. GPX8 is the GPXs. of non selenocysteine, and the selenocysteine containing GPXs can reduce H2O2 by catalyzing the reduced glutathione (GSH) to reduce the oxidative stress of the disease. The non selenocysteine GPXs, which is different from the selenocysteine containing GPXs, lacks the GSH binding domain. Therefore, they are lacking in the GSH binding domain. The mechanism that causes peroxidase activity is still controversial. Reactive oxygen species (ROS), which includes oxygen ions and peroxide and other active oxygen molecules, is an intrinsic product of the body's normal metabolism. Some scholars suggest that oxidative stress reaction is related to the occurrence and development of tumor. The so-called oxidative stress reaction refers to the body suffered. In the case of harmful stimulation, the ROS in the body produces too much to make the body oxidize beyond the oxide clearance, which causes the tissue damage caused by the imbalance of the oxidation system and the antioxidant system, including the active oxygen containing molecules of O2-, H2O2, HO2-, -OH and so on. Long-term persistent oxidative stress leads to autoimmune abnormalities, tumorigenesis, diabetes, obesity, and nerves. A variety of diseases such as degeneration and aging. The main physiological function of the thyroid gland is the synthesis of thyroid hormones. The H2O2, the key enzyme of the thyroid hormone synthesis, is essential to the thyroid peroxidase, which plays an unusually important role in the synthesis of thyroid hormones and the functioning of normal thyroid function. When the body is abnormal, H2O2 Excessive accumulation in the thyroid gland causes the thyroid to continue to expose to a high concentration of H2O2 and its incidental effect in the ROS environment, that is, oxidative stress in the oxidative stress response, causing oxidative DNA damage or hypomethylation of the thyroid cells. A large number of ROS have been confirmed to promote the proliferation, invasion, metastasis and angiogenesis of the cells of the cells. And it can escape the occurrence of apoptosis. Therefore, the abnormality of redox reaction may be associated with the occurrence of PTC, and the development of.Gpx7 is one of the members of the GPxs family, which is mainly involved in maintaining the redox homeostasis of the body. In recent years, the abnormal expression of gpx7 has been found to be associated with the development of a variety of tumors, including tubular adenocarcinoma, breast cancer, hepatocellular carcinoma and so on. However, the study of gpx7 in thyroid cancer has not yet been reported. It is known that nf- kappa B is an important nuclear transcription factor that exists in eukaryotic cells to control DNA transcription. It has a multidirectional transcriptional regulation. It has been confirmed that nf- kappa B is associated with the development of a variety of malignant tumors. It is reported that the role of gpx7 in the esophageal adenocarcinoma (EAC) is possible. It is one of the indicators for detecting cell proliferation activity by nf- kappa B signaling pathway, which is one of the indicators for detecting cell proliferation activity. It is reported that CyclinD1 is an important downstream target gene of nf- kappa bp65. The study of the relationship between gpx7 in thyroid papillary carcinoma and nf- kappa B, CyclinD1 expression has not been reported. Therefore, this study aims to study the purpose of this study. The expression of gpx7 in PTC, as well as the relationship with PTC clinicopathological parameters, and the role of gpx7 in the development of PTC and its possible mechanism are discussed, respectively, to further study the mechanism of gpx7 in the thyroid papillary carcinoma. Objective: 1, the study of gpx7 in PTC from the tissue and cell levels. The effect of expression and the effect on the proliferation and apoptosis of PTC cells and the role of gpx7 in the development of PTC,.2, the relationship between gpx7 and nf- kappa bp65, CyclinD1 expression from tissue and cell levels, and the possible mechanism of gpx7 participating in the regulation of proliferation and apoptosis of PTC cells, and the study of the gpx7 protein in the thyroid gland with Hashimoto's thyroiditis The expression in the tissue of papillary carcinoma (ptc+ht) and the influence of the inflammatory microenvironment of Hashimoto's thyroiditis on the expression of gpx7 in the thyroid papillary carcinoma are established. Methods: 1, the expression of gpx7 protein in 30 cases of PTC and 14 cases of nodular goiter (nail) was detected by immunohistochemistry. The relationship between gpx7 and clinicopathological features was analyzed, and.2 was used to construct the gpx7 gene to interfere with the lentivirus vector. After the target was validated by the Westernblot method, a successful gpx7 gene was used to infect the thyroid papillary carcinoma (K1) cells of the thyroid carcinoma (K1) cells, and the gene knockout effect was detected by the formula of qt-pcr, and a variety of cell functional experiments were used. (celigo cell count, flow cytometry, caspase3/7 detection, MTT detection, and cell clone formation experiment) to observe the proliferation, apoptosis, and clone formation of gpx7 gene knockout and non gene knockout K1 cells,.3, 30 cases of PTC, 14 cases of nodular goiter (thyroid gland), nf- kappa bp65, Cycli, and Cycli. The relationship between the expression of ND1 protein and the expression of gpx7 was analyzed.4, K1 cells were infected with the gpx7 gene to interfere with the lentivirus vector, and the nf- kappa bp65 before and after the gpx7 knockout was detected by Westernblot, and the expression of cyclinD1 protein was.5. 29 cases of thyroid papillary carcinoma with Hashimoto's thyroiditis were detected by immunohistochemistry (ptc+ht). The expression of gpx7 in 31 cases of thyroid papillary carcinoma (Group PTC), 16 cases of Hashimoto thyroiditis (Group HT) and 14 cases of nodular goiter (group A), combined with the clinicopathological parameters, the effects of the inflammatory microenvironment of Hashimoto's thyroiditis on the expression of gpx7 and the role of.6 in the thyroid papillary carcinoma were investigated, and the immunohistochemical evaluation standard was used. Criteria: each case was randomly observed 5 high times of visual field, according to the percentage and intensity of dyed cells. Positive cell percentage score standard: 10% is 0, 11%~25% is 1, 26%~50% is 2, 51%~75% is 3, 75% is 4; the dyeing strength score is 0, light yellow is 1, brown yellow is 2, Brown is 3. The score of positive cell rate and staining intensity score was the final score, 0 was negative (-), 1~4 was weak positive (+), 5~8 was moderately positive (+ +), 9~12 was strong positive (+ + +).7. Statistical method: statistical analysis was carried out by spss20.0 statistical software, and the data were expressed by mean number of standard deviation, and the differences were compared by independent sample t. Test, if the variance is not homogeneous, we use t 'test. Count data using examples (percentage), group comparison using chi square test. Correlation analysis using Pearson correlation analysis.P0.05 for difference is statistically significant. Results: 1, gpx7 on thyroid papillary carcinoma cell proliferation, apoptosis effect study (1) immunohistochemical results: (1) gpx7 expression The positive rate of.Gpx7 expression in PTC group was 100% in the cytoplasm of thyroid gland. The positive rate of gpx7 expression in group a group was 30%, and the expression of gpx7 in group PTC was significantly higher than that in group A (P0.05). (2) the expression of gpx7 was related to the maximum diameter of the tumor (r=0.601, P0.05), and the maximum diameter of the gpx7 high expression group was (2). 1.56 + 0.56cm) was significantly greater than that of gpx7 low expression group (0.56 + 0.13cm) (P0.05). (2) cell experimental results: (1) the results of two human thyroid papillary carcinoma cell lines TPC-1 and k1.qt-pcr showed that the gpx7 gene had high expression abundance in TPC-1 and K1 cells. Compared with K1 cells, the expression of K1 cells was higher, so K1 cells were selected for subsequent experimental studies (2) after the preparation of the gpx7 gene interfering lentivirus vector, the expression of gpx7 was obviously reduced after the gpx7 gene knockout, which indicated that the target had a knockout effect on the exogenous expression of the gpx7 gene and was an effective target. (3) the gpx7 gene interfered with the lentivirus vector infected K1 cells and observed under the common microscope and the fluorescence microscope. It was found that the cell infection efficiency was above 80% and the cell state was normal. The expression of gpx7 gene in K1 cells was inhibited and the knock reduction efficiency reached 81.5% after the shRNA lentivirus infection was confirmed by qPCR. (4) the results of celigo cell count showed G. The number of cell proliferation in px7 gene knockout group was significantly reduced, and the proliferation rate was only (5 + 0.32) times more than that in first days (5 + 0.32). Compared with the control group (8.44 + 0.26), the proliferation rate decreased significantly. (5) flow cytometry showed that the percentage of apoptosis in gpx7 gene knockout group was (24.08 + 0.28)%, compared with the control group (4.33 + 0.15)%, the percentage of apoptosis was obvious (6) caspase3/7 activity detection showed that the caspase3/7 activity of gpx7 gene knockout group was (257.82 + 21.28), and was significantly increased compared with the control group (100 + 5.38). (7) MTT detection found that the od490 value of gpx7 gene subtraction group increased for fifth days (3.081 + 0.160) times than that of first days (3.081 + 0.160 times), compared with the control group (increased 4.897 + 0.091 times), gpx7 gene. The cell growth multiple of the knockout group decreased significantly. (8) the cell clone formation test showed that the number of gpx7 gene knockout group clones (283 + 6) compared with the control group (710 + 10), significantly reduced.2, the possible mechanism of gpx7 in thyroid papillary carcinoma (1) immunohistochemical results (1) nf- kappa bp65 expressed in the cytoplasm of thyroid cells The expression of.Ptc in group.Ptc was significantly higher than that in group.Ptc group, and the positive expression rate of nf- kappa bp65 was 83.3%. (2) the expression of CyclinD1 expressed in group.Ptc of thyroid nucleus was significantly higher than that in group.Ptc group, and the positive rate of CyclinD1 was 96.7%, and (3) gpx7, nf- kappa bp65, and the expression of cyclinD1 protein showed significant. Positive correlation (r value was 0.721,0.589,0.703, P value was 0.05 respectively). (4) the positive rate of joint detection of gpx7 and nf- kappa bp65 in group PTC was 83.3%, the positive rate of joint detection with CyclinD1 was 100%, and the positive rate of the three joint detection was 83.3%. (2) cell experiment (1) the cell efficiency of the lentivirus infection order was higher, qPCR confirmed that gpx7 gene was effectively knocked down. (2) Wester Nblot results showed that the expression of nf- kappa bp65 protein was not significantly changed after knockout of the gpx7 gene of thyroid papillary carcinoma cell (K1 cell line), and the expression of cyclinD1 protein was increased. The difference was statistically significant (P0.05).3, and the effect of the inflammatory microenvironment on the expression of gpx7 in PTC (1) immunohistochemical results showed that it was accompanied by Hashimoto. The gpx7 protein expression score of thyroid papillary carcinoma (6.21 + 2.29) was lower than that of thyroid papillary carcinoma (8.52 + 2.41). The difference was statistically significant (P0.05). (2) single factor analysis found that the factors affecting the expression of gpx7 were associated with HT, tg-ab, TPO-Ab, tumor location and membrane invasion, among which gpx7 was associated with HT, tg-ab and TPO-Ab levels Negative correlation and positive correlation with tumor location and membrane invasion. (3) multiple factor linear regression analysis found that the factors affecting gpx7 expression were combined ht. (4) clinicopathological features, and that PTC tumors with HT inflammatory environment were mostly limited to unilateral and low incidence of membrane invasion. Conclusion: 1, immunohistochemical and cell experimental results showed GPX7 The high expression of.2 in thyroid papillary carcinoma shows that GPX7 has the role of promoting the proliferation of thyroid papillary carcinoma cells and reducing apoptosis,.3, GPX7, NF- kappa B p65, and the expression of Cyclin D1 protein in thyroid papillary carcinoma is positively correlated, suggesting the occurrence of three cases of thyroid papillary carcinoma and the possible development of thyroid papillary carcinoma. There was no change in the expression of NF- kappa B p65 protein after.4 knockdown, suggesting that the role of GPX7 in papillary thyroid carcinoma is related to NF- NF- B signaling pathway, GPX7

【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R736.1

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