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

【摘要】：第一章鼻咽癌細(xì)胞中LMP1的表達(dá)與其對TRAIL敏感性的關(guān)系 目的：有研究表明不同鼻咽癌細(xì)胞對TRAIL的敏感性不同,因此本章我們檢測對比不同鼻咽癌細(xì)胞中EB病毒潛伏膜蛋白LMP1表達(dá)水平和其對TRAIL的敏感性,并探討兩者之間的關(guān)系。 方法：采用MTT和流式細(xì)胞術(shù)檢測三種不同鼻咽癌細(xì)胞株CNE-1CNE-2、C666-1對不同濃度及不同作用時相TRAIL處理的敏感度；另外,運(yùn)用RT-PCR及Western blot檢測LMP1mRNA及蛋白在三種鼻咽癌細(xì)胞中的表達(dá)。 結(jié)果：TRAIL對三種鼻咽癌細(xì)胞起生長抑制和凋亡誘導(dǎo)作用,CNE-1對TRAIL的敏感性最高,而C666-1對TRAIL的敏感性最低。CNE-1、 CNE-2、C666-1細(xì)胞LMP1mRNA相對表達(dá)量分別為0.27±0.02、0.52±0.02、1.44±0.12,LMP1蛋白相對表達(dá)量分別為0.29±0.02、0.49±0.02、0.95±0.06。CNE-1細(xì)胞LMP1mRNA及蛋白表達(dá)量最低,而C666-1細(xì)胞LMP1mRNA及蛋白表達(dá)量最高。 結(jié)論：TRAIL能夠誘導(dǎo)鼻咽癌細(xì)胞發(fā)生凋亡,但不同鼻咽癌細(xì)胞對TRAIL的敏感性與LMP1表達(dá)存在負(fù)相關(guān),LMP1表達(dá)越高,對TRAIL的敏感性就越低。提示LMP1是影響TRAIL敏感性的抗凋亡因子。 第二章LMP1誘導(dǎo)鼻咽癌細(xì)胞發(fā)生TRAIL抵抗的體外實(shí)驗(yàn) 目的：研究表明LMP1對鼻咽癌細(xì)胞凋亡具有調(diào)控作用,且上一部分實(shí)驗(yàn)表明不同鼻咽癌細(xì)胞對TRAIL的敏感性與LMP1表達(dá)存在負(fù)相關(guān)。因此,該部分我們上調(diào)LMP1在鼻咽癌細(xì)胞中的表達(dá),并觀察LMP1基因過表達(dá)后對TRAIL凋亡誘導(dǎo)作用和凋亡信號傳導(dǎo)的影響。 方法：利用脂質(zhì)體介導(dǎo)的pGL6-LMP1上調(diào)LMP1低表達(dá)的TRAIL抵抗性鼻咽癌細(xì)胞CNE-1中LMP1的表達(dá),熒光顯微鏡檢測轉(zhuǎn)染效率；G418篩選LMP1基因穩(wěn)定表達(dá)的CNE-1細(xì)胞,RT-PCR及western blot技術(shù)檢測LMP1基因的過表達(dá)效果；通過MTT法、流式細(xì)胞術(shù)觀察LMP1過表達(dá)后對CNE-1細(xì)胞TRAIL敏感性的影響；流式細(xì)胞術(shù)、western blot、線粒體膜電位檢測觀察LMP1過表達(dá)后對死亡受體表達(dá)、細(xì)胞內(nèi)外凋亡信號通路激活、線粒體膜電位改變的影響。 結(jié)果：脂質(zhì)體介導(dǎo)的pGL6-LMP1成功上調(diào)了CNE-1細(xì)胞中LMP1的表達(dá),構(gòu)建LMP1穩(wěn)定表達(dá)細(xì)胞CNE-1-LMP1。CNE-1、轉(zhuǎn)染空白質(zhì)細(xì)胞CNE-1-pGL6、CNE-1-LMP1中LMP1mRNA相對表達(dá)量分別為,LMP1蛋白相對表達(dá)量分別為0.23±0.01、0.22±0.01、0.69±0.01。與CNE-1及轉(zhuǎn)染空白質(zhì)細(xì)胞CNE-1-pGL6比較,TRAIL對CNE-1-LMP1的細(xì)胞殺傷作用和凋亡誘導(dǎo)作用明顯減弱。死亡受體熒光標(biāo)記后流式細(xì)胞術(shù)檢測發(fā)現(xiàn)CNE-1和CNE-1-LMP1之間細(xì)胞膜蛋白DR4、DR5表達(dá)無統(tǒng)計(jì)學(xué)差異(P0.05)。Western blot結(jié)果顯示TRAIL(100ng/ml)分別作用2、4、6、12小時后,CNE-1細(xì)胞中caspase-8p43/p41, p18業(yè)單位蛋白和caspase-3p17, p10亞單位蛋白農(nóng)達(dá)明顯高于CNE-1-LMP1細(xì)胞,而tBid和caspase-9p35亞單位蛋白表達(dá)無明顯改變,且兩個細(xì)胞株間也無明顯區(qū)別。JC-1線粒體膜電位檢測法結(jié)果顯示TRAIL干預(yù)后,線粒體膜電位無明顯改變,且兩個細(xì)胞株間也無明顯區(qū)別。 結(jié)論：LMP1過表達(dá)抑制TRAIL對鼻咽癌細(xì)胞的凋亡誘導(dǎo)作用和其細(xì)胞外凋亡信號的傳導(dǎo),提示LMP1是通過抑制細(xì)胞外信號通路激活誘導(dǎo)鼻咽癌細(xì)胞發(fā)生TRAIL抵抗。 第三章LMP1通過激活PI3K/Akt通路誘導(dǎo)鼻咽癌細(xì)胞發(fā)生TRAIL抵抗 目的：研究表明PI3K/Akt通路活化是細(xì)胞發(fā)生TRAIL抵抗的重要機(jī)制,因此本部分從體外實(shí)驗(yàn)證實(shí)鼻咽癌細(xì)胞中LMP1過表達(dá)可以激活PI3K/Akt信號通路,并進(jìn)一步驗(yàn)證LMP1是通過PI3K/Akt信號通路誘導(dǎo)鼻咽癌細(xì)胞發(fā)生TRAIL抵抗。 方法：運(yùn)用western blot檢測LMP1過表達(dá)后Akt和磷酸化Akt(p-Akt)的表達(dá)改變；免疫熒光雙標(biāo)記檢測LMP1過表達(dá)后LMP1和p-Akt的共定位。選用P13K特異性抑制劑LY294002抑制PI3K/Akt信號通路后,MTT及流式細(xì)胞術(shù)檢測CNE-1、CNE-1-LMP1對TRAIL敏感性的改變；Western blot檢測細(xì)胞外信號通路的激活情況。 結(jié)果：CNE-1-LMP1細(xì)胞p-Akt蛋白表達(dá)明顯高于CNE-1細(xì)胞。免疫熒光雙標(biāo)記顯示與LMP1過表達(dá)后,p-Akt表達(dá)增高,且出現(xiàn)由胞漿向胞膜轉(zhuǎn)位,與LMP1共定位于細(xì)胞膜上。1μmol/ml LY294002預(yù)處理8小時后的CNE-1和CNE-1-LMP1中p-Akt表達(dá)明顯降低,且兩細(xì)胞間p-Akt表達(dá)無統(tǒng)計(jì)學(xué)差異(P0.05)。MTT及流式細(xì)胞術(shù)結(jié)果顯示LY294002預(yù)處理后,CNE-1、CNE-1-LMP1對TRAIL的敏感性明顯提高,且兩細(xì)胞間無明顯差異。Western blot結(jié)果顯示TRAIL(100ng/ml)分別作用2、4、6、12小時后,LY294002預(yù)處理后的CNE-1-LMP1細(xì)胞中caspase-8p43/p41, p18亞單位蛋白和caspase-3p17,p10亞單位蛋白表達(dá)明顯高于未經(jīng)LY294002預(yù)處理的CNE-1-LMP1細(xì)胞。 結(jié)論：LMP1是通過激活PI3K/Akt信號通路誘導(dǎo)鼻咽癌細(xì)胞發(fā)生TRAIL抵抗,提示針對PI3K/Akt信號通路的鼻咽癌靶向治療方案可能逆轉(zhuǎn)LMP1誘導(dǎo)的TRAIL抵抗 第四章LMP1誘導(dǎo)鼻咽癌發(fā)生TRAIL抵抗在的體內(nèi)實(shí)驗(yàn)研究 目的：進(jìn)一步從體內(nèi)實(shí)驗(yàn)證實(shí)細(xì)胞水平的實(shí)驗(yàn)結(jié)果,并利用所獲得的移植瘤組織標(biāo)本對LMP1誘導(dǎo)鼻咽癌細(xì)胞發(fā)生TRAIL抵抗的作用機(jī)制進(jìn)行初步探討。 方法：采用CNE-1和CNE-1-LMP1細(xì)胞構(gòu)建LMP1不同表達(dá)水平的鼻咽癌動物移植瘤模型,并進(jìn)行TRAIL干預(yù)。通過測量瘤體大小、HE染色驗(yàn)證其成瘤及瘤體生長情況；Western blot技術(shù)檢測移植瘤標(biāo)本中LMP1的表達(dá)情況；TUNEL熒光凋亡檢測法檢測瘤體中凋亡細(xì)胞量；計(jì)算對比TRAIL對兩種細(xì)胞腫瘤的生長抑制率。 結(jié)果：采用CNE-1和CNE-1-LMP1細(xì)胞成功構(gòu)建LMP1高表達(dá)及低表達(dá)鼻咽癌動物移植瘤模型,HE染色證實(shí)成瘤率達(dá)100%,westernblot檢測顯示CNE-1-LMP1細(xì)胞移植瘤LMP1蛋白表達(dá)高于CNE-1細(xì)胞移植瘤。CNE-1對照組移植瘤和CNE-1-LMP1對照組移植瘤在各個時間點(diǎn)腫瘤體積、處死裸鼠后腫瘤重量、TUNEL熒光凋亡檢測腫瘤細(xì)胞凋亡指數(shù)五統(tǒng)計(jì)學(xué)差異。TRAIL干預(yù)組移植瘤在各個時間點(diǎn)腫瘤體積、處死裸鼠后腫瘤重量均低于對應(yīng)對照組移植瘤,而TUNEL熒光凋亡檢測腫瘤細(xì)胞凋亡指數(shù)高于對應(yīng)對照組移植瘤。CNE-1TRAIL.干預(yù)組移植瘤在各個時間點(diǎn)腫瘤體積、處死裸鼠后腫瘤重量均低于CNE-1-LMP1移植瘤,而TUNEL熒光凋亡檢測腫瘤細(xì)胞凋亡指數(shù)高于CNE-1-LMP1移植瘤。在種瘤后各個時間點(diǎn),TRAIL對CNE-1移植瘤生長抑制率高于對CNE-1-LMP1移植瘤生長抑制率。 結(jié)論：TRAIL可以在體內(nèi)環(huán)境誘導(dǎo)鼻咽癌細(xì)胞發(fā)生凋亡,從而抑制鼻咽癌腫瘤生長,而LMP1可以在體內(nèi)環(huán)境誘導(dǎo)鼻咽癌細(xì)胞發(fā)生TRAIL抵抗。
[Abstract]:The relationship between expression of LMP1 and its sensitivity to TRAIL in nasopharyngeal carcinoma

Objective : To study the sensitivity of different nasopharyngeal carcinoma cells ( NPC ) to TRAIL , so we detected the level of latent membrane protein LMP1 in nasopharyngeal carcinoma cells and its sensitivity to TRAIL , and discussed the relationship between them .

Methods : MTT assay and flow cytometry were used to detect the sensitivity of CNE - 1CNE - 2 , C666 - 1 to different concentrations of CNE - 1CNE - 2 and C666 - 1 .
In addition , the expression of LMP1mRNA and protein in three nasopharyngeal carcinoma cells was detected by RT - PCR and Western blot .

Results : TRAIL has the highest sensitivity to TRAIL . The relative expression of CNE - 1 , CNE - 2 , C666 - 1 cells is 0.27 鹵 0.02 , 0.52 鹵 0.02 , 1.44 鹵 0.12 , respectively . The relative expression of CNE - 1 , CNE - 2 and C666 - 1 cells is 0.29 鹵 0.02 , 0.49 鹵 0.02 , 0.95 鹵 0.06 . The expression of LMP1mRNA and protein in CNE - 1 cells is the lowest , while the expression of LMP1mRNA and protein in C666 - 1 cells is the highest .

Conclusion : TRAIL can induce apoptosis of nasopharyngeal carcinoma cells , but the sensitivity of different NPC cells to TRAIL is negatively correlated with LMP1 expression . The higher the expression of LMP1 , the lower the sensitivity to TRAIL .

Chapter II In Vitro Experiment of TRAIL Resistance Induced by LMP1 in Nasopharyngeal Carcinoma

Objective : To study the effect of LMP1 on the apoptosis of nasopharyngeal carcinoma cells . The results showed that the sensitivity of LMP1 to TRAIL was negatively correlated with the expression of LMP1 . Therefore , we raised the expression of LMP1 in nasopharyngeal carcinoma cells and observed the effects of LMP1 gene overexpression on TRAIL apoptosis and apoptosis signal transduction .

Methods : The expression of LMP1 in nasopharyngeal carcinoma cell CNE - 1 with low expression of LMP1 was regulated by liposome - mediated pGL6 - LMP1 , and the transfection efficiency was detected by fluorescence microscope .
The expression of LMP1 gene was detected by G418 selection . RT - PCR and western blot were used to detect the overexpression of LMP1 gene .
The effect of LMP1 overexpression on TRAIL sensitivity of CNE - 1 cells was observed by MTT assay .
Flow cytometry , western blot and mitochondrial membrane potential were used to detect the effects of LMP1 overexpression on the expression of death receptor , the activation of apoptosis signal pathway , and the changes of mitochondrial membrane potential .

Compared with CNE - 1 and CNE - 1 - pGL6 , the expression of caspase - 8p43 / p41 , p18 and caspase - 3p17 in CNE - 1 and CNE - 1 - LMP1 were significantly higher than that of CNE - 1 and CNE - 1 - LMP1 .

Conclusion : LMP1 overexpression inhibits TRAIL ' s apoptosis - inducing effect on nasopharyngeal carcinoma cells and the conduction of extracellular apoptotic signals , suggesting that LMP1 is a potent inhibitor of TRAIL resistance in nasopharyngeal carcinoma cells by inhibiting the activation of extracellular signal pathways .

In chapter 3 , LMP1 induces TRAIL resistance in nasopharyngeal carcinoma cells by activating the 3 - 3 / 3 / 3 / 3 / 3 / 3 pathway .

Objective : To study the mechanism of inhibition of TRAIL resistance in nasopharyngeal carcinoma ( NPC ) cells . Therefore , in vitro experiments , LMP1 overexpression in nasopharyngeal carcinoma cells can be activated by the expression of LMP1 in nasopharyngeal carcinoma cells , and LMP1 is further verified to induce TRAIL resistance in nasopharyngeal carcinoma cells .

Methods : Western blot was used to detect the change of the expression of phosphorylation and phosphorylation protein in LMP1 post - expression after overexpression of LMP1 .
The sensitivity of CNE - 1 and CNE - 1 - LMP1 in CNE - 1 and CNE - 1 - LMP1 was determined by MTT assay and flow cytometry after the inhibition of the signaling pathway of PI13K .
Western blot was used to detect the activation of extracellular signal pathway .

Results : In CNE - 1 - LMP1 , CNE - 1 and CNE - 1 - LMP1 were significantly higher in CNE - 1 and CNE - 1 - LMP1 than that of CNE - 1 and CNE - 1 - LMP1 after pretreatment with LMP1 . The expression of caspase - 8p43 / p41 , p18 subunit and caspase - 3p17 in CNE - 1 and CNE - 1 - LMP1 were significantly higher in CNE - 1 and CNE - 1 - LMP1 after pretreatment with LMP1 .

Conclusion : LMP1 may induce TRAIL resistance in nasopharyngeal carcinoma cells by activation of the 3 - K / 3 - 3 signaling pathway . It is suggested that the targeted therapy for NPC may reverse the TRAIL resistance induced by LMP1 .

In Vivo Experimental Study of TRAIL Resistance Induced by LMP1 in Nasopharyngeal Carcinoma

Objective : To further investigate the experimental results of cell level in vivo and to investigate the mechanism of LMP1 - induced TRAIL resistance in nasopharyngeal carcinoma cells .

Methods : CNE - 1 and CNE - 1 - LMP1 cells were used to construct tumor models of nasopharyngeal carcinoma ( NPC ) with different expression levels .
The expression of LMP1 was detected by Western blot .
TUNEL was used to detect the amount of apoptotic cells in tumor cells .
The inhibitory rate of TRAIL on the growth of two cell tumors was calculated .

Results : CNE - 1 and CNE - 1 - LMP1 cells successfully constructed LMP1 high expression and low expression nasopharyngeal carcinoma transplanted tumor model . The tumor weight of CNE - 1 - LMP1 cells was higher than that in CNE - 1 - LMP1 group . The tumor weight of CNE - 1 - LMP1 was higher than that of CNE - 1 - LMP1 .

Conclusion : TRAIL can induce apoptosis of nasopharyngeal carcinoma cells in vivo and inhibit the growth of nasopharyngeal carcinoma . LMP1 can induce TRAIL resistance in nasopharyngeal carcinoma cells in vivo .
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R739.63

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