辐射诱导鼻咽癌细胞系凋亡及其相关基因的研究

目的Ｘ线诱导人鼻咽癌细胞系ＣＮＥ和ＣＮＥ－２凋亡及其相关基因的研究。方法应用ＤＮＡ荧光染料Ｈｏｅｃｈｓｔ３３３４２、免疫组化、ＲＴ－ＰＣＲ、ＤＮＡ杂交等方法进行观察和检验。结果一定剂量照射后,ＣＮＥ凋亡指数与时间、剂量有相关性,ＣＮＥ和ＣＮＥ－２两种细胞系存在明显不同的凋亡反应。免疫组化方法检测,ＣＮＥ的ｂｃｌ－２蛋白呈强阳性,而ＣＮＥ－２为阴性;进一步采用ＲＴ－ＰＣＲ方法检测,发现ＣＮＥ－２存在ｐ５３ｍＲＮＡ扩增产物,而ＣＮＥ却不存在。ＣＮＥ和ＣＮＥ－２的ｐ５３及ｂｃｌ－２基因的ＤＮＡ杂交结果均为阳性,但ＣＮＥ细胞ｐ５３基因的２．８ｋｂ片段较５．６ｋｂ片段明显减弱,说明在ＣＮＥ中存在ｐ５３基因的部分缺失。结论人鼻咽癌细胞系的凋亡指数有时间、剂量相关性;ＣＮＥ、ＣＮＥ－２两种细胞系的凋亡指数有明显差异,可能与ＣＮＥ中抗凋亡基因ｂｃｌ－２的过度表达及ｐ５３基因部分缺失密切相关。     

鼻咽癌中p53蛋白积聚对瘤细胞有丝分裂和凋亡的影响

目的：观察疗前鼻咽癌组织中Ｐ５３蛋白的积聚及其对瘤细胞有丝分裂和凋亡的影响。方法随机收集１９９７年疗前鼻咽癌活检标本４３例,采用免疫组化ＬＳＡＢ法ＤＯ－７一抗检测Ｐ５３蛋白的表达。在Ｈ＆Ｅ染色切片上位细胞死亡试剂盒检测瘤细胞凋亡,平均每个高倍视野下的凋亡瘤细胞数为凋亡指数（ＴＵＮＥＬ ｉｎｄｅｘ,Ｔ１）。比较高于位细胞死亡试剂盒检测瘤细胞凋亡,平均每个高倍视野下的细胞瘤细胞数为凋亡指数（ＴＵＮＥＬ     

Effect of 9-cis-retinoic acid on oral squamous cell carcinoma cell lines

Epstein–Barr virus (EBV) has been well documented in the aetiology of nasopharyngeal carcinoma (NPC), although its role as well as the genetic basis in the genesis of NPC have not been elucidated. The p53 gene mutations are infrequently found in NPC, but the expression of p53 protein, as well as bcl-2 oncoprotein, has been reported in a high percentage of cases, and also in association with EBV. Proliferating cell nuclear antigen (PCNA) has also been shown to be increased in NPC, suggesting its association among the overexpression of p53 and bcl-2 oncoprotein. We undertook this study to evaluate the correlation among these abnormalities in the development of NPC. The expression of p53 protein, bcl-2 oncoprotein, and the level of PCNA were investigated by immunohistochemistry in 53 patients with NPC. Twenty tissue samples from these patients were studied for p53 gene mutations by single strand conformation polymorphism (SSCP) and DNA sequencing as well as EBV genomes by polymerase chain reaction. Among the 53 specimens, 42 (79%) showed expression of p53 protein and 40 (75%) gave positive result for bcl-2 oncoprotein. A significant association was found between p53 expression and bcl-2 oncoprotein (P=0.002; Fisher's exact test) with 68% of the patients showing coexpression of both markers. The PCNA labelling index in the 53 patients varied from 5% to 80%. High PCNA labelling index was frequently found in the patients with overexpression of p53 protein and bcl-2 oncoprotein. The PCNA index in patients with p53 expression was significant higher than in those without p53 expression (P=0.002). Of the 20 patients, p53 mutations were found in four cases. EBV genomes were detected in 14 cases of which 12 cases showed overexpression of both p53 and bcl-2 and one case with only p53 expression and one case with bcl-2 expression. EBV genomes were detected in two cases with p53 mutations. We conclude that EBV is the important etiologic factor in NPC which may be involved in p53 and bcl-2 overexpression. The mutant p53 protein is correlated to deregulation of PCNA. p53 mutations participate in a small proportion of the tumorigenesis.     

EMP INDUCES APOPTOSIS IN HUMAN LUNG CARCINOMA CELL LINE GLC-82

The possibility of health risks resulting from exposure to electric and magnetic fields provides a strong motivation to determine how such fields interact with cells. The short, intense electromagnetic pulse (EMP) produced by high-altitude nuclear explosions may radiate over many hundreds of miles. Basically, EMP consists of a pulse of radio-frequency waves with a nearly instantaneous rise in the electric and magnetic fields and a subsequent decline in the fields. EMP radiation may be repre…     

不同p53功能状态鼻咽癌细胞株的放射生物学特性

目的探讨鼻咽癌细胞不同p53功能状态及其放射生物学特性之间的关系。方法采用脂质体介导的转染方法将携带野生型p53基因的真核表达质粒pC53-SN3，空载体质粒pCMV-NeoBarn分别转染到鼻咽癌细胞CNE1、DNE2中。p53功能检测技术明确细胞转染p53基因前后的p53功能状态。成克隆实验测定细胞存活分数。采用单击多靶模型和线性二次函数模型拟合细胞存活曲线，求出放射生物学参数Do、Dq、N和α、β、α/β、SF2值。结果转染野生型p53基因后鼻咽癌细胞获得正常p53功能。CNE1-pNeo和CNE1-wtp53细胞的％值分别为1．17、1．08Gv；Dq值分别为2．25、1．21Gy；Q值分别为0．13、0．29Gy^-1；SF2值分别为0．765、0．326。CNF2-pNeo和CNE2-wtp53细胞的Do值分别为0、92、0．84Gy；Dq值分别为1．45、1．04Gy；α值分别为0、13、0．76Gy^-1；SF2值分别为0．675、0．156。CNE1、CNE2细胞转染野生型p53基因后，Do、Dq、SF2值均减小，α值增大。结论野生型p53基因转染可以提高p53功能缺陷的鼻咽癌细胞的放射敏感性。     

自发性细胞凋亡及bcl-2和bax基因与鼻咽癌放射敏感性关系的探讨

目的探讨自发性细胞凋亡及其相关基因bcl-2和bax与鼻咽癌放射敏感性的关系。方法采用TdT酶介导的生物素化dUTP缺口末端标记技术(terminal oxynucleotidyl transferase medi-ated dUTP biotin nick end labeling,TUNEL)和免疫组化法,分别检测51例放疗前的鼻咽癌活检组织中自发性凋亡指数(spontaneous apoptotic index,SAI)和bcl-2、bax基因的表达。结果鼻咽癌活检组织均检测到细胞凋亡,SAI平均为31.19±33.83;SAI与bcl-2和bax表达均无明显相关性,但与bcl-2/bax比率呈负相关(P<0.05);SAI与鼻咽癌放射敏感性明显相关,完全消退组比残留组SAI高(P<0.05);bcl-2的表达与鼻咽癌放射敏感性呈负相关(P<0.05),而bax的表达与鼻咽癌放射敏感性无关(P>0.05);bcl-2/bax的比率与鼻咽癌放射敏感性呈负相关(P<0.05)。结论SAI、bcl-2和bcl-2/bax的比率是反映鼻咽癌放射敏感性和预测鼻咽癌放疗效果的重要指标,而bax不能单独作为判断鼻咽癌放射敏感性的指标。     

Effects of p53 overexpression on neoplastic cell mitosis and apoptosis in nasopharyngeal carcinoma

p53 gene alteration is an important molecular event in tumourigenesis of many malignancies.[1] However, the mutation rate of p53 gene in nasopharyngeal carcinoma (NPC) is lower.[2] The PCR-SSCP performed by FU Mao-fu et al. in the authors' lab in 1994 demonstrated that there were only 4 specimens showing aberrant shift in 18 NPCs (22.2%)--one in exon 5, three in exon 8.[3] On another aspect, the p53 overexpression rate almost reached about 80%.[4,5] It is well known that because the half…     

p53/bcl-2与放疗诱导的宫颈癌细胞凋亡关系的研究

目的探讨宫颈癌放疗过程中,放疗诱导的细胞凋亡与p53、bcl-2的相关性.方法选择未经治疗的宫颈癌病人20例为实验对象,搜集分割放疗前后宫颈癌组织标本,用TDT-mediated dUTP-biotin nick end labeling(TUNEL)方法检测凋亡细胞;采用单克隆抗体免疫组化ABC法检测细胞凋亡相关基因p53、bcl-2的蛋白表达水平.结果(1)在宫颈癌放疗前后,细胞凋亡阳性率和平均凋亡指数分别为25%和0.11%、75%和2.8%,放疗前后有显著差异(P＜0.001);(2)放疗后p53蛋白表达显著减少,bcl-2蛋白无显著变化;(3)放疗前后,p53基因的表达与细胞凋亡呈有意义的相关性变化.结论放射治疗诱导了宫颈癌细胞凋亡的发生,并与凋亡调节基因p53及其表达呈间接有关.     

EFFECT OF ADENOVIRUS—MEDIATED p53 GENE TRANSFER ON APOPTOSIS AND RADIOSENSITIVITY OF HUMAN

To evaluate the effect of adenovirus-mediated p53 gene(Adp53) on apoptosis and radiosensitivity of human gastric carcinoma cell lines.Methods:Recombinant adenovirus expressing wild-type p53 lines with different p53 genetic status.p53 protein expression was detected by immunohistochemistry assay and western blot assay.Cell survival was assessed using a clonogenic assay.TUNEL assay was used in determination of apoptosis.Four human gastric carcinoma cells infected with Adp53 were irradiated with 4Gy and cell cycle distribution and Sub-G1 peak were assayed by flow cytometry.Results:G2/M arrest,apoptosis and inhibition of tumor cell proliferation were induced by infection at Adp53 at 100 MOI which caused high transfer rate of wild-type p53 and strong expression of p53 protein in four human gastric carcinoma cells.The radio-enhancement ratio of Adp53 at 4Gy were3.0 for W cell,3.6 for M cell,2.2 for neo cell and 2.5 for 823 cell in vitro.Conclusion :This study demonstrated that Adp53 transfer increased cellular apoptosis and radiosensitivity of human gastric carcinoma cell lines in vitro independently on cellular intrinsic p53 status thus supporting the combination of p53 gene therapy with radiotherapy in clinical trials.     

鼻咽癌细胞凋亡及凋亡相关基因的表达

目的　探讨细胞凋亡在鼻咽癌 (NPC)中的作用。方法　采用TdT酶介导的生物素化dutp缺口末端标记技术 (Tunel)和免疫组化S P法 ,分别检测 2 0例正常鼻咽粘膜组织及 73例NPC组织的细胞凋亡率和p16、bcl 2基因的表达。结果　正常鼻咽粘膜细胞凋亡率显著高于NPC(P <0 .0 1) ,p16、bcl 2在NPC组织中的表达分别为 2 8.8% (2 1/73)、81.2 % (5 9/73) ,在正常粘膜的表达分别为 90 0 % (18/2 0 )、15 0 % (3/2 0 )。NPC中bcl 2表达与癌细胞凋亡率呈负相关 ,而p16则与凋亡率呈正相关 (P <0 .0 1)。NPC的 3年生存率资料表明 ,细胞凋亡率 >2 0、bcl 2表达阴性、p16表达阳性的患者预后较佳。结论　在NPC中 ,癌细胞凋亡明显受到抑制 ,其发生可能与bcl 2及p16基因调控障碍有关。NPC细胞凋亡与患者的预后相关。     

Frequent and variable abnormalities in p14 tumor suppressor gene in glioma cell lines

Ten glioma cell lines were examined for abnormalities of exon 1β of the p14 gene and then for abnormalities of the entire p14 gene with the use of previous findings of other exons. Abnormalities of exon 1β and the entire p14 gene were detected in eight of ten cases: homozygous deletion of the entire gene in six cases, hemizygous deletion of exon 1β with homozygous deletion of downstream exons in one case, and hemizygous deletion of the entire coding region with a missense mutation (A97V) at the C-terminal nucleolar localization domain in one case. The remaining two cases revealed tno such abnormalities. p14 gene expression was observed in the latter two cases and one case with A97V mutation in the hemizygously deleted coding region, but not in the others, including one case with only exon 1β. In the three cases with p14 gene expression, immunocytochemistry revealed p14 nucleolar staining, suggesting the retention of the functional activity of p14 protein and, in the case with the A97V mutation, an insufficient mutational effect as well. The present findings of the frequent and variable p14 gene abnormalities, including rare-type ones with or without sufficient mutational effect in glioma cell lines, might be of value for better understanding of the p14 gene and its related pathways in glioma carcinogenesis.     

食管鳞癌自发细胞凋亡和核增殖抗原p53关系的研究

目的通过对食管鳞癌组织中凋亡细胞的原位观察和核增殖抗原（ＰＣＮＡ）的表达状态的研究,探讨不同增殖情况的食管鳞癌组织细胞凋亡情况,同时研究ｐ５３基因的突变和ｐ５３蛋白的表达对食管鳞癌组织细胞凋亡的影响。方法３０例术前未经任何治疗的食管鳞癌手术标本,分别进行凋亡细胞的原位检测（ＴＵＮＥＬ）、ＰＣＮＡ、ｐ５３蛋白的免疫组化染色和ｐ５３基因５,６,７,８外显子ＰＣＲ－ＳＳＣＰ检测。结果不同增殖能力的癌组织中,细胞凋亡的程度差异有显著性（Ｐ＜０．０５）。ｐ５３基因突变组中,增殖程度高与增殖程度低者之间癌细胞凋亡的程度差异有显著性（Ｐ＜０．０５）。ｐ５３基因突变组与非突变组、ｐ５３蛋白表达阳性组与非阳性组癌细胞凋亡的程度差异无显著性（Ｐ＞０．０５）。结论食管鳞癌组织中,细胞增殖活跃则自发性凋亡也相应增多     

Neoplastic cell apoptosis in nude mice transplants with nasopharyngeal carcinoma cell lines

We know that tumor growth speed is criticallyinfluenced by the ratio of neoplastic cell proliferation andcell death, and there are two patterns of cell death, namely,necrosis and apoptosis. What is the proportion of necrosisand apoptosis seen in nude mice transplants ofnasopharyngeal carcinoma (NPC) cell lines, CNE-1 andCNE-2? Does the apoptosis play an important role inneoplastic cell death? If so, what is the pathway ofapoptosis developed in those transplants?MATERIALS AND METHODS…     

外源p53对人肺癌细胞生长的抑制

目的　观察外源性野生型p53对有p53基因突变的人肺癌细胞系生长的影响。方法　用PCR SSCP及DNA测序 ,选择p53突变的人肺巨细胞癌系 80 1 D。构建野生型p53表达质粒PZiP p53。用基因枪介导外源基因。经G41 8筛选得到转染细胞系 80 1 D p53。用PCR检测外源基因 ,观察转染细胞恶性生长的变化。结果　转染细胞系 80 1 D p53体外长期传代有外源性p53基因存在 ,转染细胞生长明显受到抑制 ,集落形成抑制率达 96% ,裸鼠异种移植致瘤性降低 ,肿瘤生长明显缓慢。结论　外源性野生型p53经基因枪导入有p53基因突变的人肺癌细胞后可长期存在于转染细胞中 ,且明显抑制所转染的癌细胞的恶性生长。     

非甾体消炎药诱导胃癌细胞凋亡及其机制的研究

目的：明确非甾体消炎药(NSAIDs)能否诱导胃癌细胞凋亡；明确不同的p53基因表型对NSAIDs诱导的细胞凋亡是否有影响；明确NSAIDs对细胞凋亡相关基因Bcl-2及Bax表达的调控。方法：通过MTT比色法检测NSAIDs对细胞生长活力的影响；应用丫啶橙(AO)染色、Annexin-V／PI双染色、共聚焦显微镜、流式细胞术检测细胞凋亡；应用RT-PCR、Western-blot方法检测bcl-2、bax基因及蛋白水平的改变。结果：NSAIDs药物吲哚美辛(Indo)和阿司匹林(Asp)对胃癌细胞株AGS(p53 ／ )、MKN28(p53-／-)均有显著的生长抑制作用，且呈时间／浓度依赖性增强；在相同作用条件下，AGS细胞的凋亡率明显高于MKN28细胞，处理组MKN28细胞凋亡数量虽有所增多，但与正常对照组相比不具有统计学意义；随着药物作用时间的延长，Bcl-2基因mRNA表达逐渐减弱，Bax基因及蛋白表达逐渐增强，在药物作用6～24小时改变最为明显。结论：一定浓度的NSAIDs作用一定时间后，可诱导胃癌细胞凋亡，这为NSAIDs的抗肿瘤应用增加了理论依据；NSAIDs不能诱导p53基因突变的MKN28胃癌细胞株发生显著的凋亡，p53基因突变可能阻断了NSAIDs的凋亡诱导效应；NSAIDs可能通过调控Bcl-2、Bax的基因及蛋白水平而诱导肿瘤细胞凋亡。     

三氧化二砷诱导鼻咽癌CNE1细胞凋亡及其作用机制的实验研究

 目的研究三氧化二砷（As₂O₃）诱导人鼻咽癌细胞株凋亡作用及其相关机制。方法采用流式细胞术，电镜、TUNEL方法检测As₂O₃诱导CNE1细胞凋亡作用，免疫组织化学法检测As₂O₃对CNE1细胞p53、bcl-2和bax蛋白表达的影响。结果经As₂O₃处理的CNE1细胞发生凋亡，表现为FCM可检测到“亚二倍体峰”，形态学上可见核固缩，染色质边集，凋亡小体形成等改变；TUNEL法可检测到细胞内呈棕色颗粒的凋亡细胞，随着药物浓度升高，凋亡细胞发生率逐渐增多，As₂O₃目浓度为0．5mg／L、1．0mg／L和2．0mg／L作用48h后，CNE1细胞的凋亡指数分别为2．66±0．64、8．15±0．96和11．59±0．68，显著高于非药物处理组（0．43±0．43，P〈0．05）。经As2O3处理的CNE1细胞内p53和bax蛋白表达较对照组明显增加，与凋亡指数呈正相关关系（r=0．554，P=0．011；r=0．891，P=0．000…）；p53和bax蛋白表达也呈正相关关系（r=0．626，P=0．003）。结论As₂O₃在体外可诱导鼻咽癌CNE1细胞凋亡，其机制可能与上调p53、bax基因表达有关。     

高能电磁脉冲诱导肺癌细胞株GLC-82凋亡的研究

背景与目的:电磁脉冲(electromagneticpulse,EMP)辐射已用于饮食行业的灭菌,且较2450MHz连续微波辐射消毒更加有效。本研究探讨高能电磁脉冲对肺癌细胞GLC-82凋亡的影响,以发现治疗肿瘤的新手段。方法:以场强为6×104V/m的EMP2min内辐照5次,然后采用细胞计数、MTT、流式细胞术及SP免疫组化法检测bcl-2和p53蛋白的表达,并对表达强度用CMIAS-Ⅱ图像分析仪在放大400倍条件下进行分析,通过上述方法观察EMP对肺癌细胞GLC-82的损伤作用,所有数据经SPSS8.0软件进行分析。结果:EMP可明显抑制肺癌细胞GLC-82的增殖与活力。照射后细胞的MTT光吸收值(A570)与对照组相比,在辐照后0h,1h和6h明显降低。流式细胞术证明,GLC-82细胞在辐照后6h发生明显的凋亡,凋亡率达13.38%。免疫组化的图像分析表明,照射后GLC-82细胞有不同程度的bcl-2蛋白表达的下调及p53蛋白表达的上调。结论:EMP可诱导肺癌细胞GLC-82的凋亡。bcl-2及p53蛋白参与了GLC-82细胞的凋亡过程。     

p16基因对鼻咽癌细胞系CNE-2增殖凋亡的影响

目的 研究ｐ１６基因对人鼻咽癌细胞系ＣＮＥ－２细胞增殖、凋亡的影响。方法 采用脂质体介导的基因转染方法,将野生型ｐ１６基因、空载体转入该基因突变的ＣＮＥ－２细胞中,同时设置不加任何质粒的ＣＮＥ－２细胞作为对照。结果 脂质体转染、Ｇ４１８筛选所获得的抗性克隆,经免疫组化检测证实转入ｐ１６基因的ＣＮＥ－２细胞有ｐ１６蛋白表达,细胞生长速度明显减慢;ＦＣＭ检测显示,细胞周期主要阻滞在Ｇ１期,Ｓ期细胞明显     

抑癌基因p53对人胃癌细胞系放射敏感性的作用

目的评价野生型抑癌基因（正常）ｐ５３对人胃癌细胞系放射敏感性的控制作用。方法用流式细胞仪分析４Ｇｙ照射后８和２４小时４种不同ｐ５３状态的人胃癌细胞系细胞周期分布和凋亡的反应。以４Ｇｙ细胞存活份数和１０Ｇｙ照射后的肿瘤生长曲线比较４种细胞的放射敏感性。结果照射４Ｇｙ后８小时和２４小时的ｐ５３正常的ＢＧＣ８２３细胞出现强烈的Ｇ１期阻滞（分别占原细胞总数的６７．９％和６１．１％）,比无照射的该细胞Ｇ１期比例有显著的增高（Ｐ＜０．０５）,并出现明显的预示凋亡的亚Ｇ１峰（ＳｕｂＧ１）,凋亡细胞比例分别达１３．０％和１５．３％;同样条件下其他３种ｐ５３异常的细胞Ｇ１期比例没有显著的变化（Ｐ＞０．０５）,都没有出现亚Ｇ１峰,凋亡细胞比例均为０．０％。ｐ５３正常的ＢＧＣ８２３细胞４Ｇｙ的存活份数明显低于其它３种细胞（Ｐ＜０．０５）;且细胞移植肿瘤１０Ｇｙ照射后比其它３种的生长受到更明显抑制（Ｐ＜０．０５）。结论以上的结果证实了野生型ｐ５３基因促进了照射后肿瘤细胞的Ｇ１期阻滞和凋亡,从而明显地提高肿瘤细胞的放射敏感性。     

康莱特注射液诱发肾癌细胞凋亡及p53,bcl—2表达的研究

目的;探讨康莱特注射液抗肿瘤的作用机制。方法：利用ＭＴＴ法分析康莱特肾癌细胞的抑制作用,末端脱氧核苷酰转移酶法检测细胞凋亡,免疫组织化学法分析ｐ５３和ｂｃｌ－２基因表达的影响。结果：康莱特抑制肾癌细胞的ＩＣ５０为１９．３１μｌ／ｍｌ,５μｌ／ｍｌ,和１０μｌ／ｍｌ康莱特注射液具有诱发细胞凋亡的作用,细胞凋亡分别为３１．３０％和８９．７６％,康莱特浓度继续增加时,细胞凋亡的数量反而减少,１５μdispla