EB病毒潜伏膜蛋白LMP1诱导鼻咽癌细胞发生TRAIL抵抗的体内外实验研究

发布时间：2018-05-26 14:42

本文选题：鼻咽癌 + TRAIL　；参考：《中南大学》2012年博士论文

【摘要】：第一章鼻咽癌细胞中LMP1的表达与其对TRAIL敏感性的关系目的：有研究表明不同鼻咽癌细胞对TRAIL的敏感性不同,因此本章我们检测对比不同鼻咽癌细胞中EB病毒潜伏膜蛋白LMP1表达水平和其对TRAIL的敏感性,并探讨两者之间的关系。方法：采用MTT和流式细胞术检测三种不同鼻咽癌细胞株CNE-1CNE-2、C666-1对不同浓度及不同作用时相TRAIL处理的敏感度；另外,运用RT-PCR及Western blot检测LMP1mRNA及蛋白在三种鼻咽癌细胞中的表达。结果：TRAIL对三种鼻咽癌细胞起生长抑制和凋亡诱导作用,CNE-1对TRAIL的敏感性最高,而C666-1对TRAIL的敏感性最低。CNE-1、 CNE-2、C666-1细胞LMP1mRNA相对表达量分别为0.27±0.02、0.52±0.02、1.44±0.12,LMP1蛋白相对表达量分别为0.29±0.02、0.49±0.02、0.95±0.06。CNE-1细胞LMP1mRNA及蛋白表达量最低,而C666-1细胞LMP1mRNA及蛋白表达量最高。结论：TRAIL能够诱导鼻咽癌细胞发生凋亡,但不同鼻咽癌细胞对TRAIL的敏感性与LMP1表达存在负相关,LMP1表达越高,对TRAIL的敏感性就越低。提示LMP1是影响TRAIL敏感性的抗凋亡因子。第二章LMP1诱导鼻咽癌细胞发生TRAIL抵抗的体外实验目的：研究表明LMP1对鼻咽癌细胞凋亡具有调控作用,且上一部分实验表明不同鼻咽癌细胞对TRAIL的敏感性与LMP1表达存在负相关。因此,该部分我们上调LMP1在鼻咽癌细胞中的表达,并观察LMP1基因过表达后对TRAIL凋亡诱导作用和凋亡信号传导的影响。方法：利用脂质体介导的pGL6-LMP1上调LMP1低表达的TRAIL抵抗性鼻咽癌细胞CNE-1中LMP1的表达,荧光显微镜检测转染效率；G418筛选LMP1基因稳定表达的CNE-1细胞,RT-PCR及western blot技术检测LMP1基因的过表达效果；通过MTT法、流式细胞术观察LMP1过表达后对CNE-1细胞TRAIL敏感性的影响；流式细胞术、western blot、线粒体膜电位检测观察LMP1过表达后对死亡受体表达、细胞内外凋亡信号通路激活、线粒体膜电位改变的影响。结果：脂质体介导的pGL6-LMP1成功上调了CNE-1细胞中LMP1的表达,构建LMP1稳定表达细胞CNE-1-LMP1。CNE-1、转染空白质细胞CNE-1-pGL6、CNE-1-LMP1中LMP1mRNA相对表达量分别为,LMP1蛋白相对表达量分别为0.23±0.01、0.22±0.01、0.69±0.01。与CNE-1及转染空白质细胞CNE-1-pGL6比较,TRAIL对CNE-1-LMP1的细胞杀伤作用和凋亡诱导作用明显减弱。死亡受体荧光标记后流式细胞术检测发现CNE-1和CNE-1-LMP1之间细胞膜蛋白DR4、DR5表达无统计学差异(P0.05)。Western blot结果显示TRAIL(100ng/ml)分别作用2、4、6、12小时后,CNE-1细胞中caspase-8p43/p41, p18业单位蛋白和caspase-3p17, p10亚单位蛋白农达明显高于CNE-1-LMP1细胞,而tBid和caspase-9p35亚单位蛋白表达无明显改变,且两个细胞株间也无明显区别。JC-1线粒体膜电位检测法结果显示TRAIL干预后,线粒体膜电位无明显改变,且两个细胞株间也无明显区别。结论：LMP1过表达抑制TRAIL对鼻咽癌细胞的凋亡诱导作用和其细胞外凋亡信号的传导,提示LMP1是通过抑制细胞外信号通路激活诱导鼻咽癌细胞发生TRAIL抵抗。第三章LMP1通过激活PI3K/Akt通路诱导鼻咽癌细胞发生TRAIL抵抗目的：研究表明PI3K/Akt通路活化是细胞发生TRAIL抵抗的重要机制,因此本部分从体外实验证实鼻咽癌细胞中LMP1过表达可以激活PI3K/Akt信号通路,并进一步验证LMP1是通过PI3K/Akt信号通路诱导鼻咽癌细胞发生TRAIL抵抗。方法：运用western blot检测LMP1过表达后Akt和磷酸化Akt(p-Akt)的表达改变；免疫荧光双标记检测LMP1过表达后LMP1和p-Akt的共定位。选用P13K特异性抑制剂LY294002抑制PI3K/Akt信号通路后,MTT及流式细胞术检测CNE-1、CNE-1-LMP1对TRAIL敏感性的改变；Western blot检测细胞外信号通路的激活情况。结果：CNE-1-LMP1细胞p-Akt蛋白表达明显高于CNE-1细胞。免疫荧光双标记显示与LMP1过表达后,p-Akt表达增高,且出现由胞浆向胞膜转位,与LMP1共定位于细胞膜上。1μmol/ml LY294002预处理8小时后的CNE-1和CNE-1-LMP1中p-Akt表达明显降低,且两细胞间p-Akt表达无统计学差异(P0.05)。MTT及流式细胞术结果显示LY294002预处理后,CNE-1、CNE-1-LMP1对TRAIL的敏感性明显提高,且两细胞间无明显差异。Western blot结果显示TRAIL(100ng/ml)分别作用2、4、6、12小时后,LY294002预处理后的CNE-1-LMP1细胞中caspase-8p43/p41, p18亚单位蛋白和caspase-3p17,p10亚单位蛋白表达明显高于未经LY294002预处理的CNE-1-LMP1细胞。结论：LMP1是通过激活PI3K/Akt信号通路诱导鼻咽癌细胞发生TRAIL抵抗,提示针对PI3K/Akt信号通路的鼻咽癌靶向治疗方案可能逆转LMP1诱导的TRAIL抵抗第四章LMP1诱导鼻咽癌发生TRAIL抵抗在的体内实验研究目的：进一步从体内实验证实细胞水平的实验结果,并利用所获得的移植瘤组织标本对LMP1诱导鼻咽癌细胞发生TRAIL抵抗的作用机制进行初步探讨。方法：采用CNE-1和CNE-1-LMP1细胞构建LMP1不同表达水平的鼻咽癌动物移植瘤模型,并进行TRAIL干预。通过测量瘤体大小、HE染色验证其成瘤及瘤体生长情况；Western blot技术检测移植瘤标本中LMP1的表达情况；TUNEL荧光凋亡检测法检测瘤体中凋亡细胞量；计算对比TRAIL对两种细胞肿瘤的生长抑制率。结果：采用CNE-1和CNE-1-LMP1细胞成功构建LMP1高表达及低表达鼻咽癌动物移植瘤模型,HE染色证实成瘤率达100%,westernblot检测显示CNE-1-LMP1细胞移植瘤LMP1蛋白表达高于CNE-1细胞移植瘤。CNE-1对照组移植瘤和CNE-1-LMP1对照组移植瘤在各个时间点肿瘤体积、处死裸鼠后肿瘤重量、TUNEL荧光凋亡检测肿瘤细胞凋亡指数五统计学差异。TRAIL干预组移植瘤在各个时间点肿瘤体积、处死裸鼠后肿瘤重量均低于对应对照组移植瘤,而TUNEL荧光凋亡检测肿瘤细胞凋亡指数高于对应对照组移植瘤。CNE-1TRAIL.干预组移植瘤在各个时间点肿瘤体积、处死裸鼠后肿瘤重量均低于CNE-1-LMP1移植瘤,而TUNEL荧光凋亡检测肿瘤细胞凋亡指数高于CNE-1-LMP1移植瘤。在种瘤后各个时间点,TRAIL对CNE-1移植瘤生长抑制率高于对CNE-1-LMP1移植瘤生长抑制率。结论：TRAIL可以在体内环境诱导鼻咽癌细胞发生凋亡,从而抑制鼻咽癌肿瘤生长,而LMP1可以在体内环境诱导鼻咽癌细胞发生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 卤 0.02 , 0.52 卤 0.02 , 1.44 卤 0.12 , respectively . The relative expression of CNE - 1 , CNE - 2 and C666 - 1 cells is 0.29 卤 0.02 , 0.49 卤 0.02 , 0.95 卤 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 .
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R739.63

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