鼻咽癌自发性与放射诱发细胞凋亡关系的研究

最近研究发现,细胞凋亡与肿瘤发生、消退密切相关.业已证明,放射治疗可以诱导肿瘤细胞发生凋亡.我们采用细胞凋亡原位末端标记技术,对鼻咽癌(nasophryngeal carcinoma,NPC)放疗前及放疗中的石蜡包埋组织进行检测,探讨NPC自发性及放射诱发的凋亡情况,以及两者之间的关系.     

泰素联合放射对人鼻咽癌细胞的作用

目的：研究泰素（Taxol)结合放射对人鼻咽癌细胞系（CNE）的联合作用效应。方法：用克隆形成法制作细胞存活曲线，流式细胞分析测定细胞周期分布。Tunel法作细胞凋亡测定。结果：不同浓度的Taxol与CNE细胞作用24h ,其细胞毒性呈剂量依赖性。在放射增敏实验中，100mmol/LTaxol作用24h，对CNE细胞有放射增敏作用，增敏比为1．23。同样剂量的Taxol作用同样时间，可诱导CNE细胞的G2＋M期阻滞。Tunel法测定细胞凋亡，10μmol/L Taxol作用24h，可诱导CNE细胞凋亡，X射线照射2Gy也可诱导CNE细胞凋亡，而10μmol/L Taxol与2Gy射线同时作用，诱导凋亡明显增加。结论：Taxol对CNE细胞有放射增敏作用，除自身对细胞凋亡有诱导作用外，还能增强放射对CNE细胞凋亡的诱导作用。     

抑制Polo样激酶1对鼻咽癌细胞辐射敏感性的影响

目的:探索抑制Polo样激酶1(Polo-like kinase 1,PLK1)对鼻咽癌(nasopharyngeal carcinoma,NPC)细胞CNE-1和CNE-2辐射敏感性的影响.方法:分别运用siRNA和小分子抑制剂BI2536抑制CNE-1和CNE-2细胞内PLK1的表达或磷酸化,通过MTT法检测抑制PLK1对NPC细胞增殖能力的影响,流式细胞技术检测对细胞周期和凋亡的影响,细胞免疫荧光检测对辐射后DNA损伤位点的影响,克隆形成实验及曲线拟合计算对辐射后细胞放射生物参数和放射增敏比(sensitizationenhancement ratio,SER)的影响.结果:与对照组相比,抑制PLK1可以明显抑制NPC细胞增殖,并诱导细胞发生G2-M期阻滞和有丝分裂灾难.抑制NPC细胞PLK1联合射线辐射后,NPC细胞克隆形成能力下降(CNE-1:P ＜0.05;CNE-2:P ＜0.05),且随着BI2536浓度的增大克隆形成能力下降更加明显(CNE-1:P ＜0.05;CNE-2:P ＜0.05);细胞生存分数明显降低(均P＜0.05);核中γ-H2AX位点数目明显增加(P＜0.05);细胞凋亡率显著升高(均P＜0.05);siR-PLK1转染CNE-1和CNE-2后SER分别为1.1988和1.3198,BI2536处理CNE-1和CNE-2后SER分别为1.5508和1.2028.结论:抑制PLK1可以抑制NPC细胞增殖,诱导发生细胞周期阻滞和有丝分裂灾难,并能显著提高NPC细胞辐射敏感性.     

放射诱导胃癌细胞的凋亡及其相关基因的研究

目的观察放射诱导人胃癌细胞株SGC-7901凋亡的作用，并进一步研究bcl-2基因及家族、bax基因、p53基因在此过程中的作用。方法用不同剂量的9 MeV β线照射SGC-7901细胞，观察细胞在受照后的不同时间生长情况。电子透射电镜下观察细胞形态变化。琼脂糖凝胶电泳检测DNA条带变化。流式细胞仪检测受照前后细胞周期及凋亡率的变化。免疫细胞化学法检测受照前后bcl-2、bax、p53基因的表达水平。结果单次剂量照射后，SGC-7901细胞凋亡率与时间、剂量有相关性。在放射诱导SGC-7901细胞凋亡的过程中，bcl-2基因表达水平下降，bax基因表达水平升高，而p53基因表达水平无变化。结论放射能够诱导胃癌细胞株SGC-7901凋亡，凋亡率在72h达高峰。bcl-2及bax基因参与了放射诱导凋亡的调控。细胞凋亡主要是通过p53基因非依赖途径。     

放射诱导HL-60细胞凋亡的流式细胞计量术分析

目的　定性和定量地分析放射诱导ＨＬ６０ 细胞凋亡。　方法　以人类白血病细胞系ＨＬ６０ 为材料，应用光学显微镜和电子显微镜及琼脂糖电泳定性分析６０ Ｃｏ 放射线诱导ＨＬ６０ 细胞凋亡的发生，应用ＤＮＡ流式细胞计量术和末端转移酶（ＴｄＴ） 介导的缺口末端标记法（ＴＵＮＥＬ） 的流式细胞定量检测ＨＬ６０ 细胞凋亡。　结果　（１） 放射后ＨＬ６０ 出现细胞体积缩小，染色质固缩致密化、边缘化，染色体断裂，凋亡小体和细胞膜保持完整等形态学特征。（２） 琼脂糖电泳表现为特征性ＤＮＡ“梯谱”。（３）流式细胞计量术发现放射诱导ＨＬ６０ 细胞凋亡在照射后６ｈ 已较明显，而且细胞凋亡百分比随着照射剂量增加而增加。ＴＵＮＥＬ法检测细胞凋亡百分比值比ＤＮＡ流式细胞分析的方法得到的值要小，但差异无显著意义。　结论　流式细胞计量术结合细胞形态学检查和（ 或） 琼脂糖电泳能较好、简便、迅速地分析放射诱导ＨＬ６０ 细胞凋亡     

鼻咽癌组织中bcl-2及EB病毒潜伏膜蛋白表达与放射诱发细胞凋亡的关系

目的　探讨鼻咽癌 (NPC)组织bcl 2及EB病毒潜伏膜蛋白 (LMP)表达与放射诱发细胞凋亡的关系。方法　采用免疫组化S P法及TdT酶介导的生物素化dUTP缺口末端标记技术 (TUNEL法 ) ,分别检测 35例NPC组织中bcl 2及EB病毒LMP的表达 ,以及放疗总量为 10Gy时NPC组织的细胞凋亡率 (AR )。结果　NPC组织bcl 2及LMP的表达率分别为 71.4%(2 5 /35 )、45 .7% (16 /35 ) ,AR为 (6 1.3± 11.8) %。放疗后的AR与LMP表达呈正相关 ,与bcl 2的表达呈负相关。结论　LMP有促进NPC放疗后细胞凋亡的生物学作用 ,其作用与bcl 2的表达无关 ;而bcl 2表达阴性者对放射线的敏感较阳性者强。     

放射和泰素对凋亡诱导敏感或抗拒鼠肿瘤p53表达的影响

放射和泰素对凋亡诱导敏感或抗拒鼠肿瘤ｐ５３表达的影响ＳａｉｔｏＹ，ｅｔａｌ．ＩｎｔＪＲａｄｉａｔｉｏｎＯｎｃｏｌｏｇｙＢｉｏｌＰｈｙｓ，１９９７，３８：６２３～７３１．许多研究表明泰素除了有强的抗癌活性外，还有放射增敏作用。放射增敏机理一是通过与细胞...     

不敏感肿瘤细胞GLC与其凋亡的关系

以往对肿瘤细胞群体辐射效应的评估，主要着眼于克隆源性细胞存活的观察，即通过测定细胞存活份数来评估放射反应。放射诱导凋亡对细胞存活份数降低所起的作用至今尚不清楚。作者采用人肺腺癌ＧＬＣ细胞系为材料研究放射诱导ＧＬＣ细胞凋亡与存活份数降低的关系并探讨其对放射敏感性的影响。１　材料和方法１．１　细胞培养：人肺腺癌ＧＬＣ细胞呈单层贴壁生长，培养液为含２０％胎牛血清的ＲＰＭＩ１６４０完全培养液，其中含青霉素和链霉素各０．１ｇ／Ｌ，置５％ＣＯ２的３７℃培养箱内培养。１．２　照射条件：将指数生长期细胞置于室…     

肿瘤细胞放射敏感性与放射诱导凋亡关系的研究

目的探讨肿瘤细胞放射敏感性与放射诱导凋亡的关系。方法使用^60Coγ射线对人小细胞型肺癌细胞株（NCI-H446）和人宫颈鳞癌细胞株（Siha）和人胃癌细胞株（SGC-7901）进行照射,采用克隆形成实验拟合细胞存活曲线,根据Do值、Dq值等放射生物学参数,比较肿瘤细胞的放射敏感性。琼脂糖凝胶电泳和流式细胞仪检测肿瘤细胞的凋亡,透射电镜观察凋亡特有的形态,流式细胞仪检测肿瘤细胞的凋亡率,分析3种肿瘤细胞的凋亡率与放射敏感性的关系。结果3种肿瘤细胞Do值的大小依次为NCI-H446Siha〉SGC-7901。琼脂糖凝胶电泳能够观察到细胞凋亡的特有梯状条带（DNALadder）,电镜能够观察到凋亡的特有的形态,如细胞核固缩、染色质浓缩、凋亡小体形成等,流式细胞仪检测到明显的凋亡峰。肿瘤细胞凋亡率随着照射剂量的增加而增高,呈良好的剂量-效应关系,并且3种肿瘤细胞株凋亡峰值的大小依次为NCI-H446〉Siha〉SGC-7901,与其放射敏感性高低一致。结论放射线能够诱导肿瘤细胞凋亡,凋亡率随着照射剂量的增加而增高。放射诱导的肿瘤细胞凋亡率越高,则其放射敏感性越高。肿瘤细胞凋亡率的检测对预测其放射敏感性有一定的价值。     

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。     

Increased incidence of tongue cancer after primary radiotherapy for nasopharyngeal carcinoma—the possibility of radiation carcinogenesis

The aim of this study was to define the risk of tongue and other aerodigestive tract cancers developing after primary radiation therapy for nasopharyngeal carcinoma (NPC). A cohort of 903 patients with non-disseminated NPC given radical radiotherapy between 1984 and 1989 was studied for the incidence of tongue cancer and other malignancies during follow-up. A contemporary cohort of 87 patients with tongue cancer, without a history of NPC, was studied for demographic data, cigarette smoking and alcohol consumption habits. These were then compared with all the NPC patients and with the NPC patients who later developed tongue cancers. There was a significantly increased number of tongue cancers following radiotherapy for NPC. The risk of developing tongue cancer after radiotherapy for NPC was 0.13% per patient per year. There was no increase in the number of other malignancies. The association between NPC and tongue cancer was that of a non-random temporal sequence with tongue cancers following NPC but not in the reverse order. The demographic data and smoking and alcohol consumption history of the 7 NPC patients who subsequently developed tongue cancer were significantly different from the de novo tongue cancer patient population. The absence of common aetiological factors between NPC and tongue cancer and the non-random sequence of tongue cancers occurring after NPC suggests that these seven tongue cancers could be radiation induced. The estimated radiation dose received by the part of the tongue developing cancer was substantial and significantly higher than the dose to the cancer-free tongue. An increase of tongue cancers after radiotherapy for NPC is reported and arguments are made in support of the hypothesis that these were radiation-induced malignancies. We suggest a decrease in the volume of tongue included within the planning target volume of NPC in the absence of oropharyngeal and/or parapharyngeal infiltration. Awareness of the association should make early diagnosis of this likely radiation-induced cancer possible.     

鼻咽癌组织放疗前后细胞增殖凋亡的变化

目的 研究鼻咽癌组织放疗前后细胞增殖凋亡的变化。方法 采用TdT酶介导的生物素化dutp制品末端标记技术（terminal oxynucleotidyl transferase mediated dUTP biotion mick end labeling,TUNEL）和免疫组化S－P法,分别检测60例鼻咽癌患者在放疗前、放疗第2周、第4周时细胞凋亡率（apoptosis rate,AR）和增殖细胞核抗原（proliferation cell nuclear antigen,PCNA）的表达。结果 鼻咽癌组织放疗后的AR明显高于放疗前;常规分割放疗组放疗第4周后的AR较放疗第2周时下降,而PCNA增殖指数升高（P＜0．01;加速超分割放疗组放疗第2周和放疗第4周的AR、PCNA增殖指数均无显著差异（P＞0．05）。结论 放射治疗鼻咽癌可以加速癌细胞的凋亡,鼻咽癌自发性细胞凋亡与放疗诱发的凋亡呈正相关。常规分割放疗过程中存在肿瘤细胞加速再增殖和细胞凋亡率下降,而采用超分割放疗可以减少鼻咽癌细胞加速再增殖,提高凋亡率。     

凋亡、PCNA及c-myc表达水平与鼻咽癌放射敏感性的相关性研究

目的:探讨凋亡、增殖细胞核抗原(PCNA)及c-myc之间的相互关系以及它们与放射敏感性的相关关系.方法:搜集31例鼻咽癌患者于放疗前及接受放射剂量1 520CGy后分别从鼻咽部肿物取活检.应用末端脱氧核苷酸转移酶(TdT)介导的脱氧核苷酸缺口末端标记(TUNEL)技术检测肿瘤细胞的凋亡率(AR),应用S-P免疫组化法检测PCNA、c-myc,并将它们与放射敏感性进行相关研究.结果:放疗前c-myc蛋白的表达水平与肿瘤细胞的自发性凋亡呈负相关、与PCNA呈正相关(P＜0.01);放疗后c-myc蛋白的表达水平与放射诱导凋亡呈正相关,与PCNA呈负相关(P＜0.01).肿瘤细胞放射敏感性级别与放疗前PCNA、放疗前凋亡率、放射诱导凋亡、放疗后PCNA下降幅度呈正相关(P＜0.01);而与c-myc无明显相关性(P＞0.05).结论:凋亡及PCNA与鼻咽癌的放射敏感性之间存在密切相关性,而c-mvc对凋亡及细胞增殖有调控作用.     

鼻咽癌放疗前及放疗中细胞凋亡的变化和意义

 目的　探讨鼻咽癌放疗前及放疗中细胞凋亡的变化及其意义。方法　采用TdT酶介导的生物素化dutp缺口末端标记技术(TUNEL),检测35例鼻咽癌患者放疗前及放疗中鼻咽部刷落癌细胞的细胞凋亡率(apoptosis rate,AR)。结果　鼻咽癌放疗前的AR为7.2％±2.4%;放疗量分别为10GY、30GY和60GY时的AR分别为64.3%±21.4%、57.2%±17.3%、16.2%±7.4%,明显高于放疗前(P＜0.01);放疗前的自发性细胞凋亡与放射治疗诱发的细胞凋亡呈正相关。结论　检测鼻咽癌患者刷落癌细胞的细胞凋亡率有助于判断放射敏感性。     

Differential cellular zinc levels in metastatic and primary nasopharyngeal carcinoma

Zinc levels are known to be elevated in certain cancer tissues. In this study, zinc content in metastatic and primary nasopharyngeal carcinoma (NPC) cells were quantitated by X-ray microanalysis at the ultrastructural level. Zinc levels of cancer cells derived from the cervical lymph node of a patient with metastatic carcinoma and that from the nasopharynx biopsy of another NPC patient with no clinical evidence of secondary spread, were analyzed. X-ray microanalysis revealed significantly higher cellular zinc levels in metastatic NPC cells. Zinc is a known anti-apoptotic agent and tumor response to radiotherapy is linked with apoptosis or programmed cell death. Raised zinc levels observed here could provide the biological basis for the observation of a higher percentage of distant metastasis in cervical node positive NPC patients treated by radiotherapy (the mainstay of treatment for NPC) as compared to those without regional nodal disease.     

CK、Tubulin-β和PCNA在鼻咽癌放疗后复发组织中的表达及意义

 目的 研究鼻咽癌中CK、Tubulin-β及PCNA的表达,探讨其与鼻咽癌放疗后复发和转移的可能机制。方法裸鼠体内移植瘤实验观察鼻咽癌细胞F1、S1放射治疗前后的细胞形态、体内增殖和转移能力,采用免疫组织化学SP法检测裸鼠移植瘤组织、鼻咽癌及放疗后复发组织中CK、Tubulin-β及PCNA的表达。结果放射后F1、S1裸鼠移植瘤瘤体质量分别较未放射组的F1、S1裸鼠移植瘤瘤体质量低（P<005）。各组均无肺转移,放射治疗后S1裸鼠移植瘤淋巴结转移率高于F1组,但差异无统计学意义（P>005）。未放射组F1裸鼠移植瘤组织中PCNA表达高于未放射S1裸鼠移植瘤（P<0.05）;放射治疗后S1裸鼠移植瘤中CK和PCNA表达均高于放射后的F1裸鼠移植瘤（P<0.05/0.01）。鼻咽癌放疗后复发组织中PCNA表达高于鼻咽癌组（P<0.05）。Spearman相关分析显示,鼻咽癌组织中CK与Tubulin-β、Tubulin-β与PCNA表达呈正相关关系,放疗后复发组织中CK与Tubulin-β和PCNA表达呈正相关（P<0.05）。鼻咽癌及放疗后复发组织中CK、Tubulin-β及PCNA的表达与患者年龄、性别、颈部淋巴结转移和临床分期无关。结论CK和Tubulin-β参与鼻咽癌放疗后复发的过程,并与细胞增殖关系密切,但不能独立作为鼻咽癌放疗后复发的预测指标。     

鼻咽癌患者放疗后耳聋的危险因素和临床处理

目的：了解鼻咽癌患者放疗后耳聋的影响因素探索耳聋的处理方法。方法：观察220例鼻咽癌患者放疗后耳聋的发生情况,对耳聋者鼓室行穿刺抽液。分析患者性别、年龄、鼻咽肿瘤T分期、侧壁侵犯、放疗前分泌性中耳炎等因素与放疗后出现耳聋的关系。结果：鼻咽癌患者放疗后耳聋的发生率为46．82％,放疗后6个月内发生率为52．42％。患者性别、年龄、鼻咽肿瘤T分期等因素对放疗后耳聋的发生无显著性影响（P〉0．05）,肿瘤侵犯鼻咽侧壁和放疗前有分泌性中耳炎与放疗后耳聋的发生显著相关（P〈0．01）。结论：肿瘤侵犯鼻咽侧壁和放疗前有分泌性中耳炎是放疗后耳聋的危险因素。鼻咽癌患者放疗后耳聋的处理目前比较好的是重复鼓室穿刺抽液配合适时倒佩戴助听器。     

Radiotherapy induces cell cycle arrest and cell apoptosis in nasopharyngeal carcinoma via the ATM and Smad pathways

Nasopharyngeal carcinoma (NPC) is a common malignant neoplasm of the head and neck which is harmful to human's health. Radiotherapy is commonly used in the treatment of NPC and it induces immediate cell cycle arrest and cell apoptosis. However, the mechanism remains unknown. Evidences suggested the activation of Ataxia telangiectasia mutated (ATM) pathway and Smad pathway are 2 of the important crucial mediators in the function of radiotherapy. In this study, we performed in vitro assays with human nasopharyngeal carcinoma CNE-2 cells and in vivo assays with nude mice to investigate the role of the ATM and Smad pathways in the treatment of nasopharyngeal carcinoma with radiotherapy. The results suggested that radiation induced activation of ATM pathway by inducing expression of p-ATM, p-CHK1, p-CHK2, p15 and inhibiting expression of p-Smad3. In addition, Caspase3 expression was increased while CDC25A was decreased, leading to cell cycle arrest and cell apoptosis. On the other hand, activation of Smad3 can inhibited the ATM pathway and attenuated the efficacy of radiation. In summary, we suggest that both ATM and Smad pathways contribute to the cell cycle arrest and cell apoptosis during nasopharyngeal carcinoma cells treated with radiation.     

鼻咽癌放射治疗中急性放射损伤的初步分析

目的：分析鼻咽癌放射治疗中不同的照射方法对急性放射损伤的影响。方法：自1996年8月－1999年8月共受治鼻咽癌患56例,其中耳前野加颈部切线照射21例,面颈联合野照射35例,其中后程超分割照射17例,6MV－X线照射,总剂量68－72Gy。分组观察急性放射损伤,包括血液系统、消化系统、皮肤和疼痛等。结果：三组急性放射损伤均存在显性差异。P＜0．01。结论：对于鼻咽癌面颈联合野及后程超分割照射应积极预防急性放射损伤,选择超分割照射应慎重,避免放疗中断,影响治疗效果。     

Mitochondrial DNA somatic mutations are frequent in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is one of the most common cancers in southern China, and is highly sensitive to radiotherapy. The control region (D-loop) of mtDNA is a polymorphic region in which point mutations occur frequently. In this study, point mutation and common deletion (CD) mutations were investigated in 23 samples of NPC tumor tissue and in the radiation-treated NPC cell line CNE2. Polymorphisms at 72 (7.28%, 72/988) nucleotide positions in D-loop region and 6 (0.75%, 6/795) nucleotide positions in part of the functional gene encoding regions were detected in all NPC patients. Of the detected polymorphisms, 8 are novel. These variants are nonencoding transitions, including np292T-->C , np517G-del, np16038A-->G, np513G-del, np16242C-->A, np513G-del, np16242C-->A and np15787T-->C transition. A total of 39 point mutations in the D-loop region of mtDNA were detected in 43.5% (10/23) of the NPC patients. Three point mutations in the functional gene encoding regions of mtDNA were detected in only 8.7% (2/23) of NPC patients. The effect of he mutation at np709G-->A in the 12sRNA gene is unclear, and the A-->G substitution at np15769 in the cytochrome B gene is a synonymous mutation. The C-->T substitution at np15970 in the T Psi C loop of the tRNA(pro) gene could alter the position of the proline residue. After irradiation, the survival fraction of CNE2 cells decreased as X-ray dose increased. Moreover, X-ray radiation could induce apoptosis and the CD mutation in a time- and dose-dependent manner, but did not induce mtDNA point mutations. A positive correlation between the apoptosis index and the ratio of CD/WT mtDNA was observed in irradiated CNE2 cells. Our results suggest that CD mutation induced by irradiation is one of the late events after apoptosis of the cancer cells, and the mtDNA CD mutation may associated with the susceptibility of NPC cells to IR-induced apoptosis.