本文關(guān)鍵詞： EGFR 核轉(zhuǎn)位 宮頸癌 放射治療 宮頸癌 EGFR 核轉(zhuǎn)位 輻射 EGFR 宮頸癌 核轉(zhuǎn)位 靶向治療　出處：《廣西醫(yī)科大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：第一部分EGFR核轉(zhuǎn)位對(duì)宮頸癌放化療預(yù)后的影響背景和目的:表皮生長(zhǎng)因子受體(EGFR)具有介導(dǎo)細(xì)胞表面信號(hào)級(jí)聯(lián)和轉(zhuǎn)位到細(xì)胞核調(diào)控許多細(xì)胞過(guò)程包括轉(zhuǎn)錄調(diào)節(jié)和DNA損傷的非同源末端連接修復(fù)作用。大量的研究證實(shí),EGFR與肺癌,肝癌,胃癌、頭頸部腫瘤等的發(fā)生、發(fā)展及治療預(yù)后有關(guān)。然而,宮頸癌細(xì)胞EGFR的表達(dá)與預(yù)后及對(duì)放療反應(yīng)的相關(guān)性仍然是有爭(zhēng)議或不確定的。最近的研究提示,一些腫瘤細(xì)胞核EGFR表達(dá)與放化療抵抗和預(yù)后差有關(guān)。有研究報(bào)道,放射線可誘導(dǎo)蛋白激酶C(Protein Kinase C,PKC)家族分子PKN1活化和EGFR二聚體的形成,以及活化的PKN1使EGFR近末端654位點(diǎn)的蘇氨酸磷酸化(EGFR pThr654),可能是介導(dǎo)EGFR核轉(zhuǎn)位的關(guān)鍵點(diǎn)之一。本文研究了局部中晚期宮頸癌細(xì)胞膜EGFR(m EGFR)、磷酸化PKN1(PKN1 pThr774)和EGFR pThr654的表達(dá)水平,探討EGFR核轉(zhuǎn)位對(duì)中晚期宮頸癌患者放化療的預(yù)后作用。方法:收集2007年1月至2012年12月在四川省腫瘤醫(yī)院行根治性同步放化療IIA-IIIB期(FIGO)宮頸癌初治患者129例。用免疫組化法檢測(cè)宮頸癌組織標(biāo)本中m EGFR、EGFR pThr654和PKN1 pThr774蛋白的表達(dá),分析其間表達(dá)的相關(guān)性,及其與宮頸癌患者的臨床病理因素和預(yù)后的關(guān)系。結(jié)果:顯微觀察顯示,m EGFR在細(xì)胞膜上表達(dá),PKN1 pThr774在細(xì)胞漿和細(xì)胞核內(nèi)均有表達(dá),而EGFR pThr654只在細(xì)胞核內(nèi)表達(dá);用X-tile軟件計(jì)算m EGFR、EGFR pThr654和PKN1 pThr774表達(dá)量的cut-off值,并根據(jù)cut-off值得出,在129例宮頸癌中,EGFR低表達(dá)為84例(65.1%,84/129),高表達(dá)45例(34.9%,45/129);EGFR pThr654高表達(dá)32例(24.8%,32/129),低表達(dá)組97例(75.2%,97/129);PKN1 pThr774高表達(dá)37例(28.7%,37/129),低表達(dá)92例(71.3%,92/129)。斯珀曼(Spearman)相關(guān)系數(shù)的顯著性檢驗(yàn)顯示,m EGFR與EGFR pThr654顯著正相關(guān)(r=0.256,P=0.015);PKN1 pThr774與EGFR pThr654與顯著正相關(guān)(r=0.217,P=0.04);多因素分析顯示,m EGFR(HR=28.469;95%CI=4.626-86.348;P=0.019)和EGFR pThr654(HR=45.137,95%CI=6.312-110.850;P=0.000)是宮頸癌患者無(wú)進(jìn)展生存期(PFS)獨(dú)立預(yù)后因素;也是總生存期(OS)的獨(dú)立預(yù)后因素(m EGFR:HR=32.278,95%CI=6.615-89.134;P=0.001和EGFR pThr654:HR=40.009;95%CI=4.425-100.962;P=0.000)。結(jié)論:m EGFR和核EGFR pThr654的高表達(dá)與宮頸癌初始放化療后獨(dú)立的較差的疾病特異性生存期有關(guān),提示這可能是導(dǎo)致放化療抵抗的因素之一。m EGFR、EGFR pThr654和PKN1 pThr774可以作為宮頸癌放化療敏感性及預(yù)后的預(yù)測(cè)指標(biāo)和治療靶標(biāo)。第二部分輻射對(duì)宮頸癌細(xì)胞EGFR核轉(zhuǎn)位及其相關(guān)蛋白的影響背景和目的:表皮生長(zhǎng)因子受體(EGFR)可以直接轉(zhuǎn)運(yùn)至核內(nèi),活化DNA-PK,增強(qiáng)DNA損傷的非同源末端連接(NHEJ)修復(fù),減低放療敏感性,是新近發(fā)現(xiàn)的EGFR傳遞信號(hào)的新途徑。我們先前的研究顯示宮頸癌組織中有核EGFR的表達(dá),EGFR和EGFR pThr654、PKN1 pThr774可能是EGFR核轉(zhuǎn)位通路上的相關(guān)蛋白,PDK1是PI3K/AKT通路上的重要蛋白,調(diào)節(jié)DNA-PK的活性。故檢測(cè)放療前后EGFR和EGFR pThr654、PKN1和PKN1pThr774、PDK1和PDK1 p Ser241、DNA-PK和DNA-PK pThr2609等蛋白表達(dá)量的變化,進(jìn)一步了解輻射對(duì)宮頸癌腫瘤細(xì)胞核轉(zhuǎn)運(yùn)的影響,并探討核轉(zhuǎn)運(yùn)的機(jī)制及在宮頸癌細(xì)胞輻射耐受中可能的作用。方法:C33A、Ca Ski、Hela和A431細(xì)胞采用6MV X射線進(jìn)行單次照射,于照射后10、20和40min分離提取細(xì)胞質(zhì)、細(xì)胞核蛋白,Western blot測(cè)定各細(xì)胞株EGFR、PDK1、PKN1、非同源末端連接(NHEJ)修復(fù)作用相關(guān)的DNA-PK及其相關(guān)磷酸化蛋白放療前后表達(dá)水平的變化。結(jié)果:四組細(xì)胞株照射之前核內(nèi)均有EGFR表達(dá),人宮頸癌Ca Ski、Hela,C33A細(xì)胞株,同人皮膚癌A431細(xì)胞株一樣,Ca Ski、Hela,也存在放療后核內(nèi)總EGFR及EGFR pThr654的表達(dá)量顯著增加(P0.05),放療后隨核內(nèi)EGFR的增加,DNA-PK pThr2609表達(dá)量也明顯增高(P0.05),兩者間有相關(guān)性。Ca Ski細(xì)胞內(nèi)PKN1 pThr774放療后也有表達(dá)量逐漸增加(P0.05),并與EGFR pThr654放療后變化具有相關(guān)性(P0.05)。Hela核內(nèi)的PDK1及PDK1 p Ser241也有放療后隨時(shí)間增加(P0.05),與DNA-PK pThr2609放療后變化有正相關(guān)性(P0.05)。結(jié)論:宮頸癌細(xì)胞放療后發(fā)生EGFR核轉(zhuǎn)位,核EGFR激活DNA損傷修復(fù)相關(guān)蛋白DNA-PK,可能是其介導(dǎo)Ca Ski、Hela,C33A細(xì)胞株放射抵抗的機(jī)制之一,并提示EGFR pThr654可能是介導(dǎo)宮頸癌細(xì)胞,無(wú)論腺癌還是鱗癌,放療后發(fā)生EGFR核轉(zhuǎn)位的關(guān)鍵點(diǎn)之一,PKN1 pThr774在Ca Ski細(xì)胞中與EGFR Thr654的磷酸化高度相關(guān)。核PDK1及其磷酸化PDK1Ser241可能與Hela細(xì)胞的放療抵抗相關(guān)。本研究還發(fā)現(xiàn)放療后多種蛋白可能發(fā)生核轉(zhuǎn)位,核轉(zhuǎn)位可能是腫瘤的一種防御機(jī)制。第三部分抑制EGFR核轉(zhuǎn)位對(duì)宮頸癌細(xì)胞輻射耐受性的研究背景和目的:前期實(shí)驗(yàn)證實(shí)宮頸癌細(xì)胞內(nèi)存在放療誘導(dǎo)的EGFR核轉(zhuǎn)位,EGFR pThr654可能是這一過(guò)程的關(guān)鍵點(diǎn),核EGFR激活DNA-PK Thr2609,可能降低放療敏感性。本實(shí)驗(yàn)探討EGFR核轉(zhuǎn)位抑制肽及EGFR靶向藥物:西妥昔單抗、吉非替尼對(duì)宮頸癌細(xì)胞輻射后核轉(zhuǎn)位的影響,及對(duì)輻射耐受性的影響。方法:采用EGFR核轉(zhuǎn)位抑制肽(p T654)及對(duì)照肽、西妥昔單抗及吉非替尼預(yù)處理人宮頸鱗癌Ca Ski、人宮頸腺癌Hela細(xì)胞16h,細(xì)胞再暴露于6MVX射線,輻射4Gy后,在0,10,20,40分不同時(shí)間點(diǎn)測(cè)定(Western blot方法)核EGFR、EGFR pThr654、以及DNA-PK和DNA-PK pThr2609的表達(dá)。用γH2AX免疫熒光法分析了西妥昔單抗、吉非替尼對(duì)輻射誘導(dǎo)的染色體損傷的影響。采用克隆形成法研究西妥昔單抗/吉非替尼是否增加宮頸癌細(xì)胞的放射敏感性,Graph Pad Prism5.0軟件擬合不同藥物組的劑量-生存曲線。采用SPSS17.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析。結(jié)果:EGFR核轉(zhuǎn)位抑制肽p T654、西妥昔單抗及吉非替尼顯著降低了放療后Ca Ski、Hela細(xì)胞核EGFR、EGFR pThr654和DNA-PK pThr2609的表達(dá)(P0.05),對(duì)照肽未有效抑制輻射引起的核轉(zhuǎn)位及相關(guān)蛋白的表達(dá),但西妥昔單抗和吉非替尼也可以誘導(dǎo)EGFR核轉(zhuǎn)位;西妥昔單抗或吉非替尼聯(lián)合放療增加了單純放療后的γ-H2AX foci數(shù)(P0.05);克隆實(shí)驗(yàn)提示西妥昔單抗聯(lián)合放療SER值為1.67,明顯增加了Ca Ski細(xì)胞的放療敏感性(P0.05);對(duì)于Hela細(xì)胞,西妥昔單抗聯(lián)合放療組、吉非替尼聯(lián)合放療組、單純放療組,SER值分別為1.015、1.009、1,均未增加Hela細(xì)胞的放療敏感性(P0.05)。結(jié)論:進(jìn)一步證實(shí)EGFR pThr654是EGFR核轉(zhuǎn)位的關(guān)鍵磷酸化位點(diǎn);抑制EGFR核轉(zhuǎn)位能提高輻射敏感性;西妥昔單抗提高Ca Ski細(xì)胞的放療敏感性,但未改善Hela細(xì)胞輻射效果,考慮兩種細(xì)胞內(nèi)信號(hào)通路可能不全相同�；A(chǔ)核EGFR高表達(dá)可能是非獲得性治療耐受的誘因,靶向藥或放療誘導(dǎo)的核轉(zhuǎn)位可能是獲得性治療耐受的誘因。
[Abstract]:The first part of the nuclear translocation of EGFR in cervical cancer chemotherapy and prognostic impact of background and purpose: the epidermal growth factor receptor (EGFR) is mediated by cell surface signaling cascade and translocation to nucleus and regulates many cellular processes including non homologous end joining repair damage and transcriptional regulation of DNA. A large number of studies confirmed that EGFR and lung cancer, liver cancer. Gastric cancer, head and neck cancer development, prognosis and treatment. However, the expression of EGFR in cervical carcinoma cells and the correlation between prognosis and response to radiotherapy is still controversial and uncertain. Recent studies suggest that some tumor cells EGFR expression is associated with resistance to chemotherapy and poor prognosis. Studies have reported that radiation the line can be induced by protein kinase C (Protein Kinase C, PKC) the formation and activation of PKN1 family molecules EGFR two dimers, and the activation of PKN1 EGFR threonine phosphorylation sites near the end of 654 (EGFR pT Hr654), may be one of the key points is mediated by the nuclear translocation of EGFR. This paper studies the local advanced cervical cancer cell membrane EGFR (m EGFR), phosphorylated PKN1 (PKN1 pThr774) expression of EGFR and pThr654, to investigate the prognostic role of chemotherapy and nuclear translocation of EGFR in patients with advanced cervical cancer. Methods: to collect from January 2007 to December 2012, radical chemoradiation IIA-IIIB in the tumor hospital of Sichuan province (FIGO) for cervical cancer patients. 129 cases were detected in cervical cancer m EGFR immunohistochemistry, the expression of EGFR pThr654 and PKN1 pThr774 protein, correlation analysis between the expression of the relationship between clinicopathological factors and prognosis of with cervical cancer patients. Results: the microscopic observation showed that the expression of M EGFR in PKN1 pThr774 in the cell membrane, cytoplasm and nucleus were expressed, while EGFR pThr654 expression only in the nucleus; calculation of M EGFR with X-tile software, EGF R pThr654 and PKN1 pThr774 expression in cut-off, and according to the cut-off value, in 129 cases of cervical carcinoma, low expression of EGFR in 84 cases (65.1%, 84/129), high expression in 45 cases (34.9%, 45/129); EGFR pThr654 high expression in 32 cases (24.8%, 32/129), 97 cases (low expression group 75.2%, 97/129); PKN1 pThr774 high expression in 37 cases (28.7%, 37/129), low expression in 92 cases (71.3%, 92/129). Shipman (Spearman) significant correlation coefficient showed a significant positive correlation between M EGFR and EGFR pThr654 (r=0.256, P=0.015); PKN1 pThr774 and EGFR pThr654 with significant positive correlation (r=0.217, P=0.04); multivariate analysis showed that m EGFR (HR=28.469; 95%CI=4.626-86.348; P=0.019) and EGFR pThr654 (HR=45.137,95%CI=6.312-110.850; P=0.000) is cervical cancer progression free survival (PFS) is an independent prognostic factor; overall survival (OS) independent prognostic factor (m EGFR:HR=32.278,95%CI=6.615-89.134; P=0.001 and EGFR PThr654:HR=40.009; 95%CI=4.425-100.962; P=0.000). Conclusion: high expression of EGFR and m in cervical cancer with initial nuclear EGFR pThr654 after radiotherapy and chemotherapy independent poor disease-specific survival, suggesting that this may be the cause of.M EGFR is one of the factors of resistance to chemotherapy, predictive biomarkers and therapeutic targets in EGFR pThr654 and PKN1 pThr774 can be used as cervical cancer chemotherapy sensitivity and prognosis. The second part radiation on human cervical cancer cells EGFR nuclear translocation and its related protein background and purpose: the epidermal growth factor receptor (EGFR) can be directly transported to the nucleus, live DNA-PK, enhance the non homologous end joining (NHEJ) DNA damage repair, reduce the radiation sensitivity, is a new way the newly discovered EGFR signal. Our previous studies showed that the expression of nuclear EGFR in cervical carcinoma, EGFR and EGFR pThr654, PKN1 pThr774 may be EGFR nuclear translocation pathway 涓婄殑鐩稿叧铔嬬櫧,PDK1鏄疨I3K/AKT閫氳礬涓婄殑閲嶈铔嬬櫧,璋冭妭DNA-PK鐨勬椿鎬，

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