本文選題：EphA2 + 鼻咽癌��； 參考：《中南大學(xué)》2012年博士論文

【摘要】：研究背景鼻咽癌(Nasopharyngeal carcinoma, NPC)是一種鼻咽上皮來源惡性腫瘤,流行于我國南方多省。目前NPC的治療以放療為主,化療是最主要的輔助治療方法。盡管放、化療技術(shù)不斷改進(jìn),但是NPC患者的療效仍改善不明顯。其中NPC細(xì)胞對(duì)化療抵抗性的產(chǎn)生是影響NPC療效的重要原因之一。因此尋找與NPC發(fā)生、發(fā)展及化療敏感性密切相關(guān)的基因,通過對(duì)該類有效基因的檢測和靶向調(diào)控,可以達(dá)到輔助診斷NPC、增加NPC的化療敏感性、改善NPC的療效、提高NPC患者的生存率等目的,具有潛在的臨床應(yīng)用價(jià)值。 促紅細(xì)胞生成素產(chǎn)生肝細(xì)胞受體A2(erythropoietin-producing hepatocellular receptor EphA2)在多種腫瘤中表達(dá)上調(diào),且其表達(dá)與腫瘤的病理分級(jí)、臨床分期、轉(zhuǎn)移、預(yù)后等密切相關(guān),參與調(diào)控腫瘤細(xì)胞的生長、增殖、遷移、侵襲、轉(zhuǎn)移等多項(xiàng)惡性生物學(xué)行為。目前,國內(nèi)外有關(guān)EphA2的研究多集中在其對(duì)腫瘤細(xì)胞侵襲轉(zhuǎn)移的影響,而EphA2對(duì)腫瘤細(xì)胞化療敏感性的調(diào)控國內(nèi)外報(bào)道甚少。紫杉醇為鼻咽癌常用化療藥物,靶向調(diào)控其化療敏感性有助于提高NPC的療效。本研究將分以下三個(gè)階段探討EphA2在NPC生長、增殖、侵襲過程中的作用,并重點(diǎn)研究EphA2對(duì)NPC紫杉醇化療敏感性的調(diào)控作用及其分子機(jī)制。 第一章EphA2在鼻咽癌中的表達(dá)及其臨床意義 目的：檢測EphA2在NPC組織和細(xì)胞株中的表達(dá)水平,探討EphA2的表達(dá)與NPC臨床病理特征的相關(guān)性。 方法：利用Western Blot技術(shù)檢測47例NPC新鮮組織標(biāo)本和21例鼻咽非腫瘤新鮮組織標(biāo)本以及NPC細(xì)胞株中EphA2蛋白的表達(dá)水平,統(tǒng)計(jì)分析EphA2的表達(dá)與NPC臨床病理特征的相關(guān)性。 結(jié)果：EphA2蛋白在NPC組織中的表達(dá)較鼻咽非腫瘤組織中的表達(dá)明顯升高,在NPC細(xì)胞株中也呈現(xiàn)高表達(dá),且EphA2蛋白的表達(dá)水平與NPC的T分級(jí)(P=0.028)、臨床分期(P=0.004)和頸部淋巴結(jié)轉(zhuǎn)移(P=0.012)密切相關(guān),組間差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論：EphA2蛋白在NPC組織和細(xì)胞中表達(dá)升高,EphA2蛋白的表達(dá)與NPC患者的T分級(jí)、臨床分期以及頸部淋巴結(jié)轉(zhuǎn)移密切相關(guān),提示EphA2在NPC的發(fā)生、發(fā)展過程中可能具有重要作用。 第二章沉默和上調(diào)EphA2的表達(dá)對(duì)鼻咽癌細(xì)胞增殖、細(xì)胞周期及侵襲能力的影響 目的：觀察調(diào)控EphA2的表達(dá)對(duì)NPC細(xì)胞的增殖、凋亡、細(xì)胞周期及侵襲能力等生物學(xué)行為的影響。 方法：利用慢病毒載體介導(dǎo)的EphA2shRNA(EphA2shRNA-LV)和EphA2基因過表達(dá)克隆(EphA2cDNA-LV)轉(zhuǎn)染NPC5-8F細(xì)胞,利用嘌呤霉素篩選建立穩(wěn)定沉默和過表達(dá)EphA2蛋白的細(xì)胞系,Western Blot驗(yàn)證EphA2蛋白沉默和上調(diào)效果。CCK-8法分別檢測EphA2沉默和過表達(dá)前后細(xì)胞的生長抑制率,流式細(xì)胞術(shù)分別檢測EphA2沉默和過表達(dá)前后細(xì)胞的凋亡率及細(xì)胞周期,Transwell侵襲實(shí)驗(yàn)檢測EphA2沉默后細(xì)胞侵襲能力的改變。 結(jié)果：1.慢病毒介導(dǎo)的RNA干擾和過表達(dá)克隆技術(shù)成功沉默和上調(diào)NPC5-8F細(xì)胞中EphA2的表達(dá),并建立了穩(wěn)定沉默和過表達(dá)EphA2的細(xì)胞系。 2.沉默EphA2蛋白的表達(dá)減弱5-8F細(xì)胞的體外增殖能力,72h開始EphA2沉默組細(xì)胞與對(duì)照組細(xì)胞生長抑制率有統(tǒng)計(jì)學(xué)差異(P0.05)；上調(diào)EphA2蛋白的表達(dá)增強(qiáng)5-8F細(xì)胞的體外增殖能力,72h始EphA2上調(diào)組細(xì)胞與對(duì)照組細(xì)胞生長抑制率比較,有統(tǒng)計(jì)學(xué)差異(P0.05)。 3.EphA2沉默組與對(duì)照組比較,G0/G1期細(xì)胞比例(%)(沉默組：74.38±3.71VS對(duì)照組：53.82±2.19)明顯增加,而S期細(xì)胞比例(沉默組：17.89±2.21VS對(duì)照組：30.41±1.55)和G2/M期細(xì)胞比例(沉默組：7.72±2.24VS對(duì)照組：15.80±3.72)則顯著降低,組間差異均具有統(tǒng)計(jì)學(xué)意義(P0.05)。上調(diào)EphA2表達(dá),G0/G1期細(xì)胞比例較對(duì)照組明顯降低(上調(diào)組47.39±3.75VS53.89±2.22),差異具有統(tǒng)計(jì)學(xué)意義(P0.05)；而S期細(xì)胞比例(38.80±7.11VS38.63±4.15)無明顯差異(P0.05),G2/M期細(xì)胞比例則較對(duì)照組增加(14.15±3.00VS7.81±2.36),差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。沉默EphA2的表達(dá),細(xì)胞的凋亡率(%)較對(duì)照組無明顯差異(P0.05),而EphA2過表達(dá)細(xì)胞的凋亡率較對(duì)照組減少(4.94±1.45VS8.99±0.84),差異具有統(tǒng)計(jì)學(xué)意義(P0.05) 4.沉默EphA2的表達(dá),5-8F細(xì)胞的體外侵襲能力較對(duì)照組明顯減弱(穿過小室到下室面的細(xì)胞數(shù)：沉默組：59±4VS對(duì)照組：103±12),差異具有顯著統(tǒng)計(jì)學(xué)意義(P0.01)。 結(jié)論：EphA2通過影響NPC細(xì)胞的周期進(jìn)展調(diào)控其體外增殖能力,并能影響NPC細(xì)胞的體外侵襲能力,提示EphA2在NPC的發(fā)展和心惡性進(jìn)展過程中發(fā)揮重要作用。 第三章EphA2對(duì)鼻咽癌細(xì)胞紫杉醇化療敏感性的調(diào)控作用及其分子機(jī)制 目的：探討EphA2對(duì)NPC細(xì)胞紫杉醇化療敏感性的影響及其分子機(jī)制。 方法：基因過表達(dá)克隆EphA2cDNA和EphA2vector質(zhì)粒瞬時(shí)轉(zhuǎn)染5-8F細(xì)胞,72h后Western Blot技術(shù)檢測EphA2的表達(dá)水平。梯度濃度紫杉醇分別作用于穩(wěn)定沉默EphA2表達(dá)的5-8FEphA2-RNAi+細(xì)胞及5-8F細(xì)胞和瞬時(shí)轉(zhuǎn)染上調(diào)EphA2表達(dá)的5-8FEphA2cDNA+細(xì)胞、5-8FEphA2vector細(xì)胞及5-8F細(xì)胞,48h后CCK-8法檢測各孔的吸光度(OD)值,計(jì)算各組細(xì)胞生存率和紫杉醇IC50濃度。IC30濃度紫杉醇分別作用于5-8FEphA2cDNA+細(xì)胞、5-8FEphA2vector組及5-8F細(xì)胞,48h后流式細(xì)胞術(shù)檢測各組細(xì)胞凋亡率和細(xì)胞周期,Western Blot技術(shù)檢測各組細(xì)胞中EphA2、細(xì)胞周期調(diào)控蛋白(CDK2、Cyclin E、p21、 p27、Rb和p-Rb)和PI3-K/Akt信號(hào)通路蛋白(Akt、p-Akt、GSK-3β、p-GSK-3p)的表達(dá)水平。以20nM、10nM和0nM濃度的P13-K/Akt抑制劑LY294002分別處理5-8FEphA2.DNA+細(xì)胞后,再予以梯度濃度紫杉醇處理,48h后CCK-8法檢測各孔吸光度(OD)值,計(jì)算各濃度抑制劑處理組細(xì)胞生存率,繪制細(xì)胞生存曲線,得出紫杉醇的IC30后,再以IC30的紫杉醇作用于以20nM、10nM和0nM濃度的P13-K/Akt抑制劑LY294002處理過的5-8FEphA2.DNA+細(xì)胞,Western Blot檢測各組EphA2.P13-K/Akt信號(hào)通路蛋白和細(xì)胞周期調(diào)控蛋白的表達(dá)水平,流式細(xì)胞術(shù)檢測各組細(xì)胞周期和凋亡率的變化。 結(jié)果：1.基因過表達(dá)克隆EphA2cDNA質(zhì)粒瞬時(shí)轉(zhuǎn)染5-8F細(xì)胞,成功上調(diào)EphA2的表達(dá),并建立了EphA2過表達(dá)的5-8F瞬轉(zhuǎn)細(xì)胞系。 2.紫杉醇作用于5-8FEphA2RNAi+細(xì)胞和5-8F細(xì)胞的IC50值分別為1.6nM和5.6nM,5-8FEphA1RNAi+細(xì)胞對(duì)紫杉醇作用的敏感性較5-8F細(xì)胞明顯增加(P0.05)；紫杉醇作用于5-8FEphA2cDNA+、5-8FEphA2vector及5-8F細(xì)胞,各組紫杉醇的IC50值分別為5nM、0.7nM、0.6nM,5-8FEphA2cDNA+細(xì)胞對(duì)紫杉醇的敏感性較5-8FEphA2vector及5-8F細(xì)胞明顯降低(P0.05)。 3.IC30紫杉醇作用于5.8FEphA2cDNA+細(xì)胞.5-8FEphA2vector細(xì)胞及5-8F細(xì)胞,流式細(xì)胞檢測各組凋亡率(%)分別為：9.84±2.08、8.66±1.59、7.74±1.34,兩兩比較,差異均無明顯統(tǒng)計(jì)學(xué)意義(PO.05).5-8FEphA2cDNA+組細(xì)胞較5-8Fvector組和5-8F組G0/G1期細(xì)胞比例明顯減少(分別為45.25±3.89VS65.85±2.28、64.52±3.31),差異具有統(tǒng)計(jì)學(xué)意義(P0.05),而S期細(xì)胞比例(分別為31.56±1.59VS25.76±1.89、24.55±3.64)和G2/M期細(xì)胞比例(分別為23.10±4.55VS8.39±0.8K10.94±3.27)則明顯增多,差異具有統(tǒng)計(jì)學(xué)意義(P0.05);5-8Fvector細(xì)胞與5-8F細(xì)胞比較,各周期細(xì)胞比例無明顯統(tǒng)計(jì)學(xué)差異(P0.05)。 4.EphA2的過表達(dá),可以抑制紫杉醇作用后的5-8F細(xì)胞中周期進(jìn)展阻滯蛋白p21和p27的表達(dá)；引起細(xì)胞周期抑制蛋白R(shí)b的表達(dá)活化、p-Rb表達(dá)水平增高；同時(shí)可以引起5-8F細(xì)胞中p-Akt表達(dá)上調(diào),而Akt的表達(dá)總量不變,但是對(duì)細(xì)胞周期啟動(dòng)蛋白CDK2和CyclinE蛋白的表達(dá)無明顯影響。 5.PI3-K/Akt抑制劑LY294002可以抑制Akt和GSK-3β的磷酸化導(dǎo)致p-Akt和p-GSK-3β的表達(dá)下調(diào),但是Akt和GSK-3β的表達(dá)總量不變；同時(shí)細(xì)胞周期進(jìn)展阻滯蛋白p21和p27的表達(dá)升高,而促細(xì)胞周期進(jìn)展蛋白p-Rb的表達(dá)水平下調(diào),細(xì)胞周期啟動(dòng)蛋白CDK2和Cyclin E的表達(dá)無明顯改變。20nM、10nM和0nM濃度的PI3-K/Akt抑制劑LY294002處理IC30濃度紫杉醇干預(yù)的5-8FEphA2cDNA+細(xì)胞,20nM和10nM抑制劑處理組細(xì)胞凋亡率分別為：9.55±0.84和9.92±2.13,較不加抑制劑(0nM)組細(xì)胞凋亡率(11.67±2.30)低,但差異無統(tǒng)計(jì)學(xué)意義(P0.05)；20nM和10nM抑制劑處理組G0/G1期細(xì)胞比例分別為：65.69±2.78、58.61±2.13,較不加抑制劑(0nM)組G0/G1期細(xì)胞比例(46.62±1.77)明顯增加,差異有統(tǒng)計(jì)學(xué)意義(P0.05),而S期細(xì)胞(20nM組：24.37±3.70,10nM組：27.08±2.19,0nM組：32.14±1.98)和G2/M期細(xì)胞(20nM組：9.94±3.97,10nM組：14.31±1.61,0nM組：21.24±1.47)則明顯增加,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。PI3-K/Akt抑制劑LY294002使5-8FEphA2cDNA+細(xì)胞對(duì)紫杉醇作用的敏感性增加。 結(jié)論：EphA2通過PI3-K/Akt/GSK-3β介導(dǎo)的信號(hào)通路影響細(xì)胞周期調(diào)控蛋白的表達(dá),進(jìn)而調(diào)控NPC細(xì)胞對(duì)紫杉醇化療的敏感性。EphA2可能作為有效的分子靶點(diǎn)應(yīng)用于調(diào)控NPC對(duì)紫杉醇化療的敏感性,對(duì)提高NPC的療效具有重要意義。
[Abstract]:Background of the Invention Nasopharyngeal carcinoma ( NPC ) is a kind of malignant tumor of nasopharyngeal epithelial origin , which is popular in many provinces in southern China . The treatment of NPC is the most important auxiliary treatment method . Although the radiotherapy and chemotherapy technique has been improved continuously , it is important to find a gene which is closely related to the occurrence , development and chemosensitivity of NPC .

The expression of EphA2 is closely related to the pathological grade , clinical stage , metastasis , prognosis and so on . At present , the study of EphA2 has little effect on tumor cell invasion and metastasis , and EphA2 plays a role in regulating the chemosensitivity of tumor cells .

The Expression of EphA2 in Nasopharyngeal Carcinoma and Its Clinical Significance

Objective : To investigate the expression level of EphA2 in NPC tissues and cell lines , and to investigate the correlation between EphA2 expression and clinical pathological characteristics of NPC .

Methods : The expression level of EphA2 protein in 47 NPC fresh tissue samples and 21 cases of nasopharyngeal non - tumor fresh tissue samples and NPC cell lines were detected by Western Blot . The correlation between EphA2 expression and clinical pathological characteristics of NPC were analyzed .

Results : The expression of EphA2 protein in NPC tissue was significantly higher than that in nasopharyngeal non - tumor tissues , and the expression level of EphA2 protein was closely related to NPC ' s T grade ( P = 0.028 ) , clinical stage ( P = 0.004 ) and cervical lymph node metastasis ( P = 0.012 ) .

Conclusion : EphA2 protein expression increases in NPC tissues and cells . EphA2 protein expression is closely related to T grade , clinical stage and cervical lymph node metastasis in NPC patients , suggesting that EphA2 may play an important role in NPC ' s development and development .

Chapter 2 silencing and up - regulation of EphA2 expression on the proliferation , cell cycle and invasion ability of nasopharyngeal carcinoma cells

Objective : To investigate the effects of EphA2 expression on the proliferation , apoptosis , cell cycle and invasion ability of NPC cells .

Methods : Using EphA2 shRNA ( EphA2shRNA - LV ) and EphA2 ( EphA2 cDNA - LV ) mediated by lentivirus vector , NPC5 - 8F cells were transfected into NPC5 - 8F cells . The inhibitory rates of EphA2 silencing and overexpression of EphA2 protein were determined by Western Blot .

Results : 1 . lentivirus - mediated RNA interference and overexpression cloning techniques successfully silenced and up - regulate EphA2 expression in NPC5 - 8F cells , and established a cell line that stabilizes silent and overexpressed EphA2 .

2 . The expression of the silent EphA2 protein attenuated the in vitro proliferation ability of 5 - 8F cells , and the growth inhibition rate of EphA2 silent group cells and control group was significantly different at 72 h ( P0.05 ) .
The increase of EphA2 protein expression enhanced the in vitro proliferation ability of 5 - 8F cells , and EphA2 up - regulated the growth inhibitory rate of EphA2 at 72h . There was statistical difference ( P0.05 ) .

3 . The percentage of cells in G0 / G1 phase ( 74.38 鹵 3.71VS control group : 53.82 鹵 2.19 ) increased significantly compared with control group , while the percentage of S phase cells ( silence group : 17.89 鹵 2.21VS control group : 30.41 鹵 1.55 ) and G2 / M phase cell ratio ( 15.80 鹵 3.72 ) decreased significantly ( P0.05 ) .
There was no significant difference in S phase cell proportion ( 38.80 鹵 7.11VS38.63 鹵 4.15 ) ( P0.05 ) . The percentage of apoptotic cells in G2 / M phase increased ( 14.15 鹵 3.00VS7.81 鹵 2.36 ) , and the difference was statistically significant ( P0.05 ) . The apoptosis rate of EphA2 overexpression cells was lower than that in control group ( 4.94 鹵 1.45V8.99 鹵 0.84 ) , and the difference was statistically significant ( P0.05 ) .

4 . The expression of EphA2 and the in vitro invasion ability of 5 - 8F cells were significantly decreased compared with those in the control group ( the number of cells passing through the chamber to the lower chamber : the silent group : 59 鹵 4VS control group : 103 鹵 12 ) , the difference was statistically significant ( P0.01 ) .

Conclusion : EphA2 regulates its in vitro proliferation ability by influencing the cycle progression of NPC cells , and can affect the in vitro invasion ability of NPC cells , suggesting that EphA2 plays an important role in the development of NPC and the progression of cardiac malignant progression .

The Effect of EphA2 on the Sensitivity of Paclitaxel Chemotherapy in Nasopharyngeal Carcinoma and Its Molecular Mechanism

Objective : To investigate the effect of EphA2 on the chemosensitivity of paclitaxel in NPC cells and its molecular mechanism .

Methods : The expression levels of EphA2 , CDK2 , Cyclin E , p21 , p27 , Rb and p - Rb , and PI3 - K / A were detected by Western Blot . The expression levels of EphA2 , CDK2 , Cyclin E , p21 , p27 , Rb and p - Rb in each group were determined by Western Blot . The expression levels of EphA2 , cyclin E , p21 , p27 , Rb and p - Rb in each group were determined by Western Blot .

Results : 1 . The gene overexpression of EphA2 cDNA plasmid transiently transfected 5 - 8F cells , successfully raised EphA2 expression , and established a 5 - 8F transient cell line of EphA2 overexpression .

2 . IC50 values of paclitaxel acting on 5 - 8FEphA2RNAi + cells and 5 - 8F cells were 1.6 nM and 5.6 nM , respectively , and the sensitivity of 5 - 8FEphA1RNAi + cells to paclitaxel increased significantly ( P0.05 ) .
The IC50 values of 5 - 8FEphA2vector and 5 - 8F cells were 5 nM , 0.7 nM , 0.6 nM , 5 - 8FEphA2cDNA + cells were significantly lower than those of 5 - 8FEphA2vector and 5 - 8F cells ( P0.05 ) .

3 . The percentage of apoptosis rate ( % ) was 9.84 鹵 2.08 , 8.66 鹵 1 . 59 , 7.74 鹵 1 . 34 in 5 - 8FEphA2vector cells and 5 - 8F cells . The percentage of cells in S phase ( 31.56 鹵 1.59VS25 . 76 鹵 1 . 89 , 24.55 鹵 3.64 ) and G _ 2 / M phase were significantly increased ( P < 0 . 05 ) .

4 . The overexpression of EphA2 can inhibit the expression of cyclin p21 and p27 in 5 - 8F cells after paclitaxel .
the expression of the cyclin Rb is activated , and the expression level of the p - Rb is increased ;
At the same time , the expression of p - in 5 - 8F cells was up - regulated , while the total amount of the expression was unchanged , but there was no significant effect on the expression of CDK2 and Cyclin E protein in the cell cycle .

5 . PI3 - K - 3.beta . phosphorylation led to downregulation of p - and p - gsk - 3.beta . , but the total amount of p3 - 3 beta was unchanged .
At the same time , the expression level of cyclin E was down regulated , and the expression level of cyclin E was down regulated . The expression of cyclin E was 9.55 鹵 0.84 and 9.92 鹵 2.13 respectively . The apoptosis rate was 11.67 鹵 2.30 , but there was no statistical significance ( P0.05 ) .
The percentage of G0 / G1 phase cells was 65.69 鹵 2.78 , 58.61 鹵 2.13 , and the percentage of G0 / G1 phase cells ( 46.62 鹵 1.77 ) increased significantly in the 20 nM and 10 nM groups ( P 0.05 ) , while the S phase cells ( 20 nM group : 24.37 鹵 3.70 , 10 nM group : 27.08 鹵 2.19 , 0nM group : 14.31 鹵 1.61 , 0nM group : 21.24 鹵 1.47 ) increased significantly ( P0.05 ) .

Conclusion : EphA2 can regulate the expression of cell cycle regulatory protein by PI3 - K - / - / - 3 - 尾 - mediated signal pathway , and then regulate the sensitivity of NPC cells to paclitaxel chemotherapy . EphA2 may be used as an effective molecular target to regulate NPC ' s sensitivity to paclitaxel chemotherapy , and it is of great significance to improve NPC ' s efficacy .

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R739.63

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