鼻咽癌细胞系SUNE中EBV—LMP1基因对永生化人胚肾上皮细胞生长 …

研究鼻咽癌细胞系ＳＵＮＥ中ＥＢ病毒潜伏膜蛋白１基因对上皮细胞生长特性的影响、探索ＬＭＰ１在鼻咽癌发生中所起的作用。方法 用ＬＭＰ１基因真核表达质粒转染人胚肾上皮细胞,检测ＬＭＰ１的表达,观察细胞的生长状态,生长速率,在软琼脂中的集落形成能力及对裸鼠的致瘤能力。     

鼻咽癌细胞系SUNE中EBV-LMP1基因对上皮细胞增殖的影响

目的研究鼻咽癌细胞系ＳＵＮＥ中ＥＢＶ┐ＬＭＰ１基因对上皮细胞增殖的影响,探索ＬＭＰ１在鼻咽癌发生中所起的作用。方法用ＬＭＰ１基因真核表达质粒转染人胚肾上皮细胞,检测ＬＭＰ１的表达,观察细胞在软琼脂中的集落形成能力,ＭＴＴ吸收能力以及ＰＣＮＡ的表达情况。结果被ＬＭＰ１基因转染的细胞生长旺盛,能在软琼脂中形成多个集落,ＭＴＴ吸收能力增强,ＰＣＮＡ的表达水平增高。结论ＬＭＰ１基因能明显改变上皮细胞的生物学行为,促进细胞的生长、增殖和转化,使转染的上皮细胞获得肿瘤细胞的生长特征。     

鼻咽癌细胞SUNE中EBV—LMP1基因对上皮细胞增殖的影响

目的 研究鼻咽癌细胞系ＳＵＮＥ中ＥＢＶ－ＬＭＰ１基因对上皮细胞增殖的影响,探索ＬＭＰ１在鼻咽癌发生中所起的作用。方法 用ＬＭＰ１基因真核表达质粒转染人胚肾上皮细胞,检测ＬＭＰ１的表达,观察细胞在软琼脂中的集落形成能力,ＭＴＴ吸收能力以及ＰＣＮＡ的表达情况。结果 被ＬＭＰ１基因转染的细胞生长旺盛,能在软琼脂中形成多个集落,ＭＴＴ吸收能力增强,ＰＣＮＡ的表达水平增高。结论 ＬＭＰ１基因能明显改变上皮细     

EB病毒的BARF1基因在人支气管上皮细胞恶性转化和肿瘤形成中的作用

目的探讨EBV-BARF1基因对人支气管上皮细胞生长特性的影响,并初步探讨其作用机制。方法构建真核重组表达载体pcDNA3-BARF1,转染人支气管上皮细胞(HBEC),检测BARF1基因的表达,观察细胞生长状况、生长速度,在软琼脂中集落形成能力及对裸鼠的致瘤能力。结果EBV-BARF1基因转染细胞生长旺盛,失去接触抑制能力,生长速度增快,在软琼脂中形成多个集落,并在裸鼠体内成瘤。结论EBV的早期基因BARF1能明显地改变上皮细胞的生物学行为,使细胞发生恶性转化、获得肿瘤细胞的生长特性,该基因不仅与上皮细胞的早期永生化有关,在细胞的恶性转化及肿瘤形成过程中亦可能起重要的作用。     

定量研究EB病毒潜在膜蛋白对人鼻咽癌细胞系CNE1的作用

目的研究ＥＢ病毒ＬＭＰ对人高分化鼻咽癌细胞株ＣＮＥ１细胞形态及ＤＮＡ含量的影响。方法采用电穿孔基因转染技术，将重组ＥＢＶ┐ＬＭＰ表达质粒转染鼻咽癌细胞株ＣＮＥ１细胞。以载体质粒转染及ＣＮＥ１细胞为对照，用免疫荧光和图像分析方法，观察细胞ＬＭＰ表达、细胞形态及ＤＮＡ含量的变化。结果ＬＭＰ表达质粒转染后细胞浆及细胞膜ＬＭＰ强阳性表达。形态定量及ＤＮＡ含量测定表明，ＬＭＰ表达后，ＣＮＥ１细胞明显变小（Ｐ＜０．０１），核浆比显著增大（Ｐ＜０．０１），核浆的变化主要由胞浆面积的增减所致，而与核面积的改变则无明显正比关系；ＤＮＡ含量明显增高（Ｐ＜０．０１／０．０５），且倍体分布更分散而右移。结论ＬＭＰ对ＣＮＥ１细胞生长有明显的促进作用，可致细胞恶性程度增高且分化受抑，有向低分化鳞癌发展倾向，提示ＬＭＰ在ＮＰＣ的发生发展过程及其演进中可能起重要作用     

EB病毒编码的BARF1基因在人上皮细胞恶性转化中的作用

背景与目的:EB病毒(Epstein-Barrvirus,EBV)的BamHⅠA区域的早期基因BARF1能使猴肾上皮细胞永生化,使人淋巴细胞和呲类动物的成纤维细胞发生恶性转化,但有关该基因在人上皮细胞肿瘤发生发展中的作用未见报道。本研究的目的是观察EBV-BARF1基因对人上皮细胞生长特性的影响及能否使之发生恶性转化。方法:构建真核重组表达载体pcDNA3-BARF1,转染人支气管上皮细胞(HBE),建立稳定表达BARF1基因的HBE细胞株;观察细胞生长状况、生长速度、在软琼脂中集落形成能力及对裸鼠的致瘤能力。结果:EBV-BARF1基因转染细胞生长旺盛,失去接触抑制能力,生长速度增快,并在裸鼠体内成瘤。结论:EBV的早期基因BARF1作为病毒的癌基因,在支气管上皮细胞的恶性转化中可能起着重要作用。     

EB病毒潜伏膜蛋白抑制鼻咽癌细胞分化的定量分析

为了探讨ＥＢ病毒潜伏膜蛋白（ＥＢＶ－ＬＭＰ）对鼻咽癌（ＮＰＣ）细胞体外分化及分化诱导剂二甲基亚砜（ＤＭＳＯ）敏感性的影响。以ＥＢＶ－ＬＭＰ基因转染人高分化鼻咽癌细胞株（ＣＮＥ１）,并用ＤＭＳＯ诱导,采用免疫组化及图像分析法观察细胞分化及形态的变化。结果显示：ＤＭＳＯ体外诱导ＣＮＥ１细胞生长明显受到抑制,细胞变大,核浆比值明显变小（Ｐ＜０．０１）,细胞角蛋白表达显著增高（Ｐ＜０．０１）;ＬＭＰ在体外可明显促进ＣＮＥ１细胞的生长,细胞变小,核浆比值明显变大（Ｐ＜０．０１）,细胞角蛋白表达显著下降（Ｐ＜０．０５）,有向低分化鳞癌发展倾向;ＬＭＰ基因转染细胞诱导后体外生长和角蛋白表达无显著改变（Ｐ＞０．０５）。结果表明,ＤＭＳＯ可在一定程度上诱导ＣＮＥ１细胞分化;ＬＭＰ对ＣＮＥ１细胞生长有明显的促进作用,可抑制细胞分化;ＬＭＰ可降低ＣＮＥ１细胞对终末分化信号的反应,这有助于对ＮＰＣ发生机理的深入研究及防治     

人NHE1 反义基因转染对SGC-7901 胃癌细胞增殖和凋亡的影响

 目的 探讨人NHE1反义基因转染对SGC-7901胃癌细胞株增殖和凋亡的影响，探索胃癌治疗的新方法。方法 采用脂质体法将构建好的人NHE1基因反义真核表达栽体转染至SGC-7901胃癌细胞中，比较观察细胞转染前后生长曲线、双层软琼脂克隆形成能力、裸鼠成瘤能力以及细胞周期和细胞凋亡率的变化。结果 NHE1反义基因能使SGC-7901胃癌细胞生长速度减慢，双层软琼脂集落形成能力、体外生长增殖能力降低，细胞凋亡率增加，裸鼠体内成瘤能力显著降低。结论 反义NHE1基因能抑制SGC-7901胃癌细胞增殖，诱导细胞凋亡，具有良好的胃癌治疗效果，提示NHE1基因可成为胃癌治疗的新靶点，具有一定的临床应用前景。     

间隙连接基因Cx43表达对肺癌细胞体内成瘤生长的抑制

目的探讨间隙连接基因表达和细胞通讯功能对肿瘤生长的抑制作用。方法以高转移性人肺癌ＰＧ细胞为材料,该细胞的间隙连接基因Ｃｘ４３表达抑制,细胞通讯功能缺陷。用Ｃｘ４３ｃＤＮＡ转染ＰＧ细胞,分离转染子克隆,与只转染空载体ｃＤＮＡ的对照组ＰＧ进行比较。用Ｎｏｒｔｈｅｒｎ分子杂交和染料传输方法检查间隙连接表达情况,并观察细胞在体外和裸鼠体内生长。结果空载体对照组与未转染组ＰＧ相似,Ｃｘ４３ｍＲＮＡ无表达,通讯功能缺陷,细胞生长快,在软琼脂内集落形成率高（１１．６％）,植入裸鼠体内２８天,平均瘤重３．４７ｇ。转染组细胞Ｃｘ４３ｍＲＮＡ表达升高,通讯功能增强,细胞生长慢,在软琼脂内集落形成率和在裸鼠体内生长速度明显低于对照组,抑制率分别为９０％和７５％。结论间隙连接基因Ｃｘ４３表达对肺癌细胞有抑瘤作用。     

BRD7基因转染对鼻咽癌细胞生长的抑制作用

目的：探讨鼻咽癌负相关基因BRD7对鼻咽癌细胞系HNE1生长的影响。方法：构建BRD7基因真核表达载体pcDNA3.1( )/BRD7重组体,采用脂质体介导转染技术,将BRD7真核表达重组粒和空载体质粒分别导入鼻咽癌细胞系HNE1,Southern杂交和RT－PCR分别检测外源性DNA的整合和BRD7基因的表达,并借助细胞生长曲线、软琼脂集落形成试验、流式细胞计数和裸鼠接种方法对转染细胞的生物学行为进行了检测。结果：转染BRD7基因的HNE1生长倍增时间为53h,较HNE1（23．9h)和空载体转染HNE1（24．1h）明显延长,流式细胞仪表明,BRD7表达升高延缓细胞由G0－G1期进入S期,BRD7转染HNE1在软琼脂中集落形成率较对照组显著下降（P＜0．01）,裸鼠接种试验显示BRD7基因转染细胞HNE1生长速度受到抑制。结论：BRD7基因重表达有助于HNE1的恶性表型的逆转;BRD7是一个鼻咽癌相关的抑瘤基因良好的候选者。     

Functional interaction of Ugene and EBV infection mediates tumorigenic effects

Epstein-Barr virus (EBV) infection is associated with many human neoplasms, in which EBV-derived latent membrane protein-1 (LMP1) appears to be critical, but its exact oncogenic mechanism remains to be defined. To this end, our initial microarray analyses identified a LMP1-inducible gene, Ugene, originally characterized as a binding partner for uracil DNA glycosylase 2, which is highly expressed in malignant colon cancer. In this report, it was found that Ugene, designated herein as LMP1-induced protein (LMPIP), was induced, in a time-dependent manner, in EBV-infected peripheral blood mononuclear cells and LMP1-transfected 293 cells. Functionally, when compared with mock-transfected cells, overexpression of LMPIP in nasopharyngeal carcinoma (NPC) cell lines resulted in a decrease in reactive oxygen species production and maintained mitochondria membrane potential (Δψ) loss induced by H(2)O(2). The NPC cells transfected with LMPIP also showed a decrease in G1 population and an increase in the cell population in sub-G1 and multiploid phase, concomitant with increased levels of cell cycle activators, including cyclin D1 and CDK4. In contrast, silencing of LMPIP expression in the NPC tumor cell lines with short hairpin RNA interference revealed significantly decreased cell population at G1/S phase, while the number of cells in multiploid phase increased. Significantly, NPC cells with LMPIP knock-down also showed a decrease in tumorigenic and transforming activity induced by ectopic LMP1 expression, as determined by analyses of soft agar foci and tumor size in nude mice. Further, elevated LMPIP expression was also noted in cytoplasm and nuclei in EBV-infected NPC tumor cell mass and non-EBV-infected tumor cell lines. These results suggested that LMPIP may have an important mediator role in EBV-mediated neoplasm and may serve as a new target for therapy of tumors induced by EBV infection.     

EB病毒潜伏膜蛋白1对鼻咽癌CNE1细胞增殖的影响

目的探讨EB病毒潜伏膜蛋白1（LMP1）对人高分化鼻咽癌CNE1细胞增殖的影响。方法采用电穿孔基因转染技术。将带有绿色荧光蛋白GFP报道系统的EB病毒LMP1基因真核表达质粒导入CNE1,以载体质粒转染及CNE1细胞为对照组,用免疫组化检测LMP1蛋白的表达;用细胞体外增殖实验检测细胞OD比值;用裸小鼠成瘤实验观察移植瘤体积倍增时间和生长率。结果内动物实验细胞移植后CNE1GL组潜伏期缩短,第7、8、9周时,其平均肿瘤体积生长显著高于对照组（P〈0.05）;体外细胞增殖实验3组细胞OD比值差异无显著性（P〉0.05）。结论EBV-LMP1对CNE1细胞的体内增殖能力可能有促进作用。     

Establishment and characterization of a novel nasopharyngeal carcinoma cell line (SUNE2) from a Cantonese patient

The undifferentiated form of nasopharyngeal carcinoma (NPC) is the most common malignant head and neck cancer in South China, especially in Cantonese populations. However, few NPC cell lines have been established from the patients in this region. In this study, we established a new NPC cell line, termed SUNE2, from a Cantonese patient with undifferentiated NPC. This cell line had extremely low concentrations of Epstein-Barr virus (EBV) DNA in long-term culture and expressed low levels of latent membrane protein 1 (LMP1), latent membrane protein 2A (LMP2A), BamH1-A right frame 1 (BARF1), EBV-encoded RNA-1 (EBER1), and EBV-encoded RNA-2 (EBER2) in early passages. SUNE2 cells also showed much stronger transforming ability than 5-8F cells in colony formation assays and anchorage- independent growth assays in soft agar, and they only need 2 weeks to form tumors in nude mice. In summary, the SUNE2 cell line is a new in vitro model that can be used for further research on the mechanisms underlying the occurrence and development of NPC.     

EBV latent membrane protein 1 effects on plakoglobin, cell growth, and migration

Latent membrane protein 1 (LMP1), the major oncoprotein of EBV, is likely responsible for many of the altered cellular growth properties in EBV-associated cancers, including nasopharyngeal carcinoma (NPC). In this study, the effects of LMP1 on cell growth and migration were studied in the context of the EBV-positive C666-1 NPC cell line. In the soft agar transformation and Transwell metastasis assays, LMP1 enhanced cell growth and migration through activation of phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor-kappaB (NF-kappaB) signaling. Inhibitors of PI3K, Akt, and NF-kappaB signaling dramatically reduced these enhanced properties. An IkappaBalpha super-repressor also blocked these effects. However, constitutive activation of Akt alone did not alter cell growth, suggesting that both PI3K/Akt and NF-kappaB activation are required by LMP1. These enhanced effects required the full-length LMP1 encompassing both the PI3K/Akt-activating COOH-terminal activation region (CTAR) 1 and the nonredundant NF-kappaB-activating regions CTAR1 and CTAR2. LMP2A, a latent protein that is also frequently expressed in NPC, similarly activates the PI3K/Akt pathway; however, its overexpression in C666-1 cells did not affect cell growth or migration. LMP1 also decreased expression of the junctional protein plakoglobin, which was shown to be partially responsible for enhanced migration induced by LMP1. This study reveals that in epithelial cells the transforming properties of LMP1 require activation of both PI3K/Akt and NF-kappaB and shows that the loss of plakoglobin expression by LMP1 is a significant factor in the enhanced migration.