本文選題：鼻咽癌 + TRAIL　； 參考：《中南大學》2012年博士論文

【摘要】：第一章鼻咽癌細胞中LMP1的表達與其對TRAIL敏感性的關系 目的：有研究表明不同鼻咽癌細胞對TRAIL的敏感性不同,因此本章我們檢測對比不同鼻咽癌細胞中EB病毒潛伏膜蛋白LMP1表達水平和其對TRAIL的敏感性,并探討兩者之間的關系。 方法：采用MTT和流式細胞術檢測三種不同鼻咽癌細胞株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,運用RT-PCR及Western blot檢測LMP1mRNA及蛋白在三種鼻咽癌細胞中的表達。 結果：TRAIL對三種鼻咽癌細胞起生長抑制和凋亡誘導作用,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細胞LMP1mRNA相對表達量分別為0.27±0.02、0.52±0.02、1.44±0.12,LMP1蛋白相對表達量分別為0.29±0.02、0.49±0.02、0.95±0.06。CNE-1細胞LMP1mRNA及蛋白表達量最低,而C666-1細胞LMP1mRNA及蛋白表達量最高。 結論：TRAIL能夠誘導鼻咽癌細胞發(fā)生凋亡,但不同鼻咽癌細胞對TRAIL的敏感性與LMP1表達存在負相關,LMP1表達越高,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誘導鼻咽癌細胞發(fā)生TRAIL抵抗的體外實驗 目的：研究表明LMP1對鼻咽癌細胞凋亡具有調控作用,且上一部分實驗表明不同鼻咽癌細胞對TRAIL的敏感性與LMP1表達存在負相關。因此,該部分我們上調LMP1在鼻咽癌細胞中的表達,并觀察LMP1基因過表達后對TRAIL凋亡誘導作用和凋亡信號傳導的影響。 方法：利用脂質體介導的pGL6-LMP1上調LMP1低表達的TRAIL抵抗性鼻咽癌細胞CNE-1中LMP1的表達,熒光顯微鏡檢測轉染效率；G418篩選LMP1基因穩(wěn)定表達的CNE-1細胞,RT-PCR及western blot技術檢測LMP1基因的過表達效果；通過MTT法、流式細胞術觀察LMP1過表達后對CNE-1細胞TRAIL敏感性的影響；流式細胞術、western blot、線粒體膜電位檢測觀察LMP1過表達后對死亡受體表達、細胞內外凋亡信號通路激活、線粒體膜電位改變的影響。 結果：脂質體介導的pGL6-LMP1成功上調了CNE-1細胞中LMP1的表達,構建LMP1穩(wěn)定表達細胞CNE-1-LMP1。CNE-1、轉染空白質細胞CNE-1-pGL6、CNE-1-LMP1中LMP1mRNA相對表達量分別為,LMP1蛋白相對表達量分別為0.23±0.01、0.22±0.01、0.69±0.01。與CNE-1及轉染空白質細胞CNE-1-pGL6比較,TRAIL對CNE-1-LMP1的細胞殺傷作用和凋亡誘導作用明顯減弱。死亡受體熒光標記后流式細胞術檢測發(fā)現(xiàn)CNE-1和CNE-1-LMP1之間細胞膜蛋白DR4、DR5表達無統(tǒng)計學差異(P0.05)。Western blot結果顯示TRAIL(100ng/ml)分別作用2、4、6、12小時后,CNE-1細胞中caspase-8p43/p41, p18業(yè)單位蛋白和caspase-3p17, p10亞單位蛋白農達明顯高于CNE-1-LMP1細胞,而tBid和caspase-9p35亞單位蛋白表達無明顯改變,且兩個細胞株間也無明顯區(qū)別。JC-1線粒體膜電位檢測法結果顯示TRAIL干預后,線粒體膜電位無明顯改變,且兩個細胞株間也無明顯區(qū)別。 結論：LMP1過表達抑制TRAIL對鼻咽癌細胞的凋亡誘導作用和其細胞外凋亡信號的傳導,提示LMP1是通過抑制細胞外信號通路激活誘導鼻咽癌細胞發(fā)生TRAIL抵抗。 第三章LMP1通過激活PI3K/Akt通路誘導鼻咽癌細胞發(fā)生TRAIL抵抗 目的：研究表明PI3K/Akt通路活化是細胞發(fā)生TRAIL抵抗的重要機制,因此本部分從體外實驗證實鼻咽癌細胞中LMP1過表達可以激活PI3K/Akt信號通路,并進一步驗證LMP1是通過PI3K/Akt信號通路誘導鼻咽癌細胞發(fā)生TRAIL抵抗。 方法：運用western blot檢測LMP1過表達后Akt和磷酸化Akt(p-Akt)的表達改變；免疫熒光雙標記檢測LMP1過表達后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及流式細胞術檢測CNE-1、CNE-1-LMP1對TRAIL敏感性的改變；Western blot檢測細胞外信號通路的激活情況。 結果：CNE-1-LMP1細胞p-Akt蛋白表達明顯高于CNE-1細胞。免疫熒光雙標記顯示與LMP1過表達后,p-Akt表達增高,且出現(xiàn)由胞漿向胞膜轉位,與LMP1共定位于細胞膜上。1μmol/ml LY294002預處理8小時后的CNE-1和CNE-1-LMP1中p-Akt表達明顯降低,且兩細胞間p-Akt表達無統(tǒng)計學差異(P0.05)。MTT及流式細胞術結果顯示LY294002預處理后,CNE-1、CNE-1-LMP1對TRAIL的敏感性明顯提高,且兩細胞間無明顯差異。Western blot結果顯示TRAIL(100ng/ml)分別作用2、4、6、12小時后,LY294002預處理后的CNE-1-LMP1細胞中caspase-8p43/p41, p18亞單位蛋白和caspase-3p17,p10亞單位蛋白表達明顯高于未經LY294002預處理的CNE-1-LMP1細胞。 結論：LMP1是通過激活PI3K/Akt信號通路誘導鼻咽癌細胞發(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LMP1誘導的TRAIL抵抗 第四章LMP1誘導鼻咽癌發(fā)生TRAIL抵抗在的體內實驗研究 目的：進一步從體內實驗證實細胞水平的實驗結果,并利用所獲得的移植瘤組織標本對LMP1誘導鼻咽癌細胞發(fā)生TRAIL抵抗的作用機制進行初步探討。 方法：采用CNE-1和CNE-1-LMP1細胞構建LMP1不同表達水平的鼻咽癌動物移植瘤模型,并進行TRAIL干預。通過測量瘤體大小、HE染色驗證其成瘤及瘤體生長情況；Western blot技術檢測移植瘤標本中LMP1的表達情況；TUNEL熒光凋亡檢測法檢測瘤體中凋亡細胞量；計算對比TRAIL對兩種細胞腫瘤的生長抑制率。 結果：采用CNE-1和CNE-1-LMP1細胞成功構建LMP1高表達及低表達鼻咽癌動物移植瘤模型,HE染色證實成瘤率達100%,westernblot檢測顯示CNE-1-LMP1細胞移植瘤LMP1蛋白表達高于CNE-1細胞移植瘤。CNE-1對照組移植瘤和CNE-1-LMP1對照組移植瘤在各個時間點腫瘤體積、處死裸鼠后腫瘤重量、TUNEL熒光凋亡檢測腫瘤細胞凋亡指數(shù)五統(tǒng)計學差異。TRAIL干預組移植瘤在各個時間點腫瘤體積、處死裸鼠后腫瘤重量均低于對應對照組移植瘤,而TUNEL熒光凋亡檢測腫瘤細胞凋亡指數(shù)高于對應對照組移植瘤。CNE-1TRAIL.干預組移植瘤在各個時間點腫瘤體積、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胞凋亡指數(shù)高于CNE-1-LMP1移植瘤。在種瘤后各個時間點,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。 結論：TRAIL可以在體內環(huán)境誘導鼻咽癌細胞發(fā)生凋亡,從而抑制鼻咽癌腫瘤生長,而LMP1可以在體內環(huán)境誘導鼻咽癌細胞發(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 .
【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R739.63

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