【作者】 姚鸿超；

【导师】 季文樾；

【作者基本信息】 中国医科大学 ， 耳鼻咽喉科学， 2007， 博士

【摘要】 肿瘤坏死因子相关凋亡诱导配体诱导喉鳞状细胞癌凋亡的实验研究实验目的喉鳞状细胞癌(LSCC)是头颈部最常见的恶性肿瘤之一，近年来其发病率有明显增长的趋势，易发生淋巴结转移，预后较差，严重威胁着人类的健康。目前临床上对于LSCC的诊断和治疗多采用联合应用手术、放疗和化疗等方法。虽然随着诊断和治疗技术的不断提高，治疗后的生存时间以及喉功能的保留都有明显提高，但对于晚期及复发患者的治疗效果仍不尽如人意。并且，喉鳞癌对大部分化疗药物不敏感，应用化疗药物常常达不到明显的治疗效果，还存在着一定的毒副作用，给治疗带来了困难。因此早期发现、早期治疗、寻找新的有效的治疗药物以及减少对正常组织的损害成为迫切需要解决的问题。近年来从研究基因改变和凋亡信号传导异常的肿瘤发生机制入手，发现通过一系列细胞因子介导的肿瘤细胞凋亡可杀灭多种肿瘤细胞，为恶性肿瘤的治疗提供了一个新的方向。凋亡(apoptosis)是细胞自身维持体内环境稳态平衡的一种程序化死亡机制，在清除肿瘤细胞、自身反应性淋巴细胞和病毒感染细胞维持机体正常生理功能中起重要作用。现代肿瘤学证明，恶性肿瘤的发生、发展是一个多因素、多阶段、多基因作用的结果，细胞凋亡在肿瘤的发生、发展中发挥着重要作用，很多基因参与了这一过程，并发挥了各自不同的作用。细胞增殖和凋亡的失衡将导致肿瘤的发生并影响肿瘤的生物学行为。肿瘤坏死因子相关诱导凋亡配体(Tumor necrosis factor related apoptosis induceligand，TRAIL)是新近发现的TNF家族新成员，它与TNF-α和Fasl一样作为凋亡细胞外活化因子与特异的受体结合而发挥作用，与前两者比较，其诱导肿瘤细胞凋亡的能力更强而没有明显的细胞毒性，抗癌谱广，即使是全身用药，也不会象TNF-α那样产生严重的炎症反应。因此，TRAIL被认为是很有潜力且有效的抗肿瘤因子，已用于恶性肿瘤治疗的研究。目前对于TRAIL抑制肿瘤的实验已经取得了一定的成绩。发现其能有效的抑制人肝癌、乳腺癌、肾癌、鼻咽癌、食管癌等肿瘤在动物体内的生长。但TRAIL在喉鳞癌中的研究尚未见报道。TRAIL及其受体在喉鳞状细胞癌中的表达分布情况、其抑制喉鳞状细胞癌的效果如何，仍有待于进一步探讨。为了进一步研究其对喉鳞状细胞癌的作用机制，我们通过逆转录-聚合酶链反应(reversetranscription-polymerase chain reaction，RT-PCR)方法以及免疫组织化学方法，检测了68例LSCC和30例正常喉黏膜中TRAIL及其死亡受体DR4、DR5，诱骗受体DcR1、DcR2的表达及分布情况；其后，应用不同浓度TRAIL作用于体外培养的喉鳞癌Hep-2细胞，通过倒置显微镜及透射电镜观察细胞的微观形态学改变，应用噻唑蓝染色(MTT)、流式细胞仪、原位末端标记(TUNEL)等方法，分析TRAIL对Hep-2细胞凋亡诱导作用的机制；并且建立人喉鳞癌裸鼠动物模型，应用TRAIL进行治疗，观察其在动物体内抑瘤效果，应用苏木精-伊红(H-E)染色及透射电镜进行肿瘤微观形态学观察，研究TRAIL抑制喉鳞癌的机制。材料和方法1、选取中国医科大学附属第一医院耳鼻咽喉科及哈尔滨医科大学附属第二医院耳鼻咽喉-头颈外科2005年3-6月期间手术切除的68例喉鳞癌标本，应用RT-PCR及免疫组化方法检测TRAIL及其受体DR4、DR5、DcR1、DcR2的表达情况，取同期手术切除的30例正常喉粘膜作为对照。2、分别将不同浓度的TRAIL加入到体外培养的喉鳞癌Hep-2细胞中，应用倒置显微镜，透射电镜观察经TRAIL作用后细胞的微观形态学改变，应用MTT法绘制细胞的生长曲线，观察细胞生长抑制率。应用流式细胞仪及TUNEL检测细胞凋亡发生率。3、应用Hep-2细胞株建立人喉鳞癌裸鼠动物模型并应用TRAIL进行治疗，观察肿瘤的生长情况，裸鼠的生存状态，以及死亡发生的情况。并分别在光学显微镜，透射电镜下对两组的肿瘤组织进行微观形态学观察。结果1、经RT-PCR及免疫组化检测发现，在LSCC及正常喉黏膜组织中普遍存在着TRAIL及其受体的表达，并且存在着表达类型的差异。其中，TRAIL及诱骗受体DcR1、DcR2在LSCC中表达较低，而在正常喉黏膜中则表达较高，差异具有统计学意义(P均＜0.01)。死亡受体DR4、DR5在LSCC及正常喉黏膜中的表达无明显差异(P均＞0.05)。2、RT-PCR结果显示，在早期喉癌(Ⅰ+Ⅱ期)，其表达TRAIL及诱骗受体DcR较高，而晚期喉癌(Ⅲ+Ⅳ)中则表达较低，差异具有统计学意义(P均＜0.05)，死亡受体DR在两组间无明显差异(P均＞0.05)；TRAIL及DcR在分化程度高的肿瘤组中表达也高，在分化程度中、低组的肿瘤表达低，差异具有统计学意义(P均＜0.01)，死亡受体DR在两组间无明显差异(P均＞0.05)。3、免疫组化分析发现，在早期喉癌(Ⅰ+Ⅱ期)，其表达TRAIL及诱骗受体DcR较高，而在晚期喉癌(Ⅲ+Ⅳ)中则表达较低，差异具有统计学意义(TRAILP＜0.05，DcR P＜0.01)，死亡受体DR在两组间无明显差异(P均＞0.05)；TRAIL及DcR在分化程度高的肿瘤组中表达高，在分化程度中、低组的肿瘤表达低，差异具有统计学意义(TRAIL P＜0.05，DcR1 P＜0.01，DcR2 P＜0.05)，死亡受体DR在两组间无明显差异(P均＞0.05)。4、通过倒置显微镜观察发现TRAIL对Hep-2细胞的作用与药物的剂量存在着明显相关系，TRAIL的浓度越高，其对细胞生长的抑制作用也越显著。5、电镜观察发现，应用TRAIL处理后的Hep-2细胞中可见细胞发生凋亡，细胞内出现核碎裂，核仁消失，染色质固缩，电子密度增高，线粒体肿胀，嵴不清，并观察到有凋亡小体形成。6、MTT证实不同浓度的TRAIL作用于体外培养的喉癌Hep-2细胞24小时，其细胞生长抑制率随着浓度的增加逐渐增大，而流式细胞仪分析，当TRAIL浓度为(1，10，100ng／ml)时，诱导凋亡率分别为11.49±0.36％，22.31±0.82％，59.64±1.10％，与对照组(TRAIL浓度0ng／ml)凋亡率3.13±0.12％之间比较，差异均具有统计学意义(P均＜0.01)。7、TUNEL检测经TRAIL作用后的所有标本都能观察到细胞核染成棕黄色的凋亡细胞。喉鳞癌Hep-2细胞经TRAIL(1，10，100ng／ml)处理后凋亡率分别为16.66±0.80％，42.01±2.58％，68.93±2.12％，与对照组(TRAIL浓度0ng／ml)凋亡率为3.32±0.19％之间比较差异均具有统计学意义(P均＜0.01)。8、喉鳞癌裸鼠动物模型均建立成功。实验发现，注射TRAIL(100ng／ml)治疗的实验组裸鼠状态良好，无明显消瘦，瘤体生长缓慢、停滞，而应用PBS的对照组肿瘤持续增长，体积明显增大，呈多中心生长，裸鼠状态差，渐消瘦，呈现恶液质状态，甚至死亡；观察期间内，对照组裸鼠死亡8只，死亡率为40％，而应用TRAIL的实验组，裸鼠仅死亡1只，死亡率为5％。治疗后两组裸鼠肿瘤体积、肿瘤重量相比较，实验组肿瘤明显小于对照组，差异具有统计学意义(P均＜0.01)，抑瘤率为53.8％。9、H-E染色光镜观察可见，对照组肿瘤细胞生长旺盛，细胞分裂相多见，萎缩坏死区较少；而TRAIL治疗组肿瘤生长受抑制，分裂不明显，出现多处萎缩坏死灶。10、电镜观察实验组肿瘤细胞生长受到抑制，核内染色质电子密度增高，胞质内膜结构减少，线粒体电子密度增大，嵴不清，核固缩，有凋亡小体形成。而对照组肿瘤细胞生长分裂旺盛，细胞间连接呈融合状态，胞质内线粒体嵴清晰，游离核蛋白体丰富，核膜结构完好。结论1、在LSCC及正常喉黏膜组织中，均存在TRAIL及其四个受体的表达，TRAIL及诱骗受体DcR在LSCC组织中与在正常喉黏膜中表达存在差异，这种分布特征是TRAIL对不同细胞表现出的选择性杀伤作用的基础。2、在LSCC组织中，TRAIL与死亡受体结合而发挥作用，诱导喉鳞癌肿瘤细胞发生凋亡，而在正常喉黏膜组织中诱骗受体可以竞争死亡受体与TRAIL结合从而抑制凋亡。3、TRAIL及诱骗受体DcR的表达分布与LSCC的病理分期以及分化程度均存在明显的相关性。4、人喉鳞癌Hep-2细胞对TRAIL的作用敏感，在体外TRAIL对Hep-2细胞的生长存在着抑制作用。其作用机制是通过诱导细胞发生凋亡来实现的。5、TRAIL诱导喉鳞癌Hep-2细胞凋亡的作用具有剂量的依赖性，增大用药剂量可使其作用增强。6、TRAIL可以通过诱导喉鳞癌肿瘤细胞凋亡从而抑制人喉鳞癌肿瘤在动物体内的生长。更多还原

【Abstract】 IntroductionThe laryngeal squamous cell carcinoma (LSCC) , with the high malignant degree, the early metastasis tendency to lymph node and a bad prognosis, is one of the most primary malignant tumor occurred in head and neck. At present, the primary treatments for LSCC include operation, radiotherapy and chemotherapy. With the development of diagnosing and treatment technique, some important progresses have been made in retention of laryngeal function, prolonging patients life after applying these treatment measures. But, however, the effect of all these measures is not as good as expected in the advanced and re-occurred cancer. Therefore, in order to improve the therapeutic effect for this type cancer, the issue need badly to be solved is to find and treat the LSCC early with new medicines and techniques to reduce the damage to the normal tissue. Recently, with the development of studying of gene transformation and apoptotic signal transduction in tumor pathogenesis, it has been discovered that apoptosis induced by anti-tumor factor may kill various tumor cells and application anti-tumor factor has acquired many exciting results in treatment of some tumors. Thus this finding may provide a new therapy for the LSCC.Apoptosis, a way of programmed cell death in our body, keep a homeostasis in balance and play an important role in cleaning tumor cells, achroacyte auto reactivity, virus infected cells. The genesis of malignant tumor is a multiple stage which involves many kinds of oncogenes and anti-oncogenes. The reasons why the tumor genesis has been formerly discussed is that un-controllable cell proliferation is the mainly cause of tumor.TRAIL (Tumor necrosis factor related apoptosis induce ligand), a new member of TNF family same as TNF-αand Fasl, plays its effect after binding with the specific receptor. Compared with Fas system, the advantage of TRAIL is that it only induces apoptosis of tumor cells without obvious toxicity to the normal cells and has been considered as an anti-tumor factor.At present, the study of TRAIL application is in exploratory stage, and the effect of TRAIL in inhibiting the growth of liver cancer, breast cancer, renal carcinoma and nasopharyngeal carcinoma has been successfully proved in nude mice experimental model. But, however, the expression of TRAIL and TRAIL-receptor in LSCC is absent and the correlation between its expression and clinical pathology is not clear. There has no report about the therapeutic effect of TRAIL in LSCC. So it is necessary to do a further study about the effect of TRAIL in treating LSCC in human.In order to illustrate the mechanism of the effect of TRAIL on LSCC, RT-PCR method (reverse transcription polymerase chain reaction) and immunohistochemistry method were used to detect the distribution and expression of TRAIL and TRAILR in 68 cases of LSCC and 30 cases of laryngeal normal tissues. In vitro the apoptosis effect induced by TRAIL in laryngeal squamous Hep-2 cells was detected by the MTT method, flow cytometry, TUNEL method and transmission electron microscope. In vivo, the nude mice model of bearing laryngocarcinoma was established by using human laryngeal squamous carcinoma cell line (Hep-2).The effect for inhibiting the LSCC was studied after given TRAIL to the animals. And also, the microstructure and ultra-micro-structural changes of carcinoma during the experiment were observed under microscope and transmission electron microscope.Materials and methods1、The 68 cases of LSCC tissue were collected from the department of otorhinolaryngology at the First Affiliated Hospital of China Medical University and Second Affiliated Hospital of Harbin Medical University during March to June 2005. RT-PCR method and Immunohistochemistry method were used to detect expression of TRAIL and TRAIL receptor DR4, DR5, DcR1, DcR2.2、Human laryngeal squamous carcinoma Hep-2 cell line was treated withdifferent concentration of TRAIL in vitro. The morphologic changes of laryngeal squamous carcinoma Hep-2 cell were observed with inverted phase contrast microscope and transmission electron microscope. The inhibition ratio of tumor cells was determined by MTT colorimetric assay, the incidence of cell apoptosis was determined by flow cytometry and TUNEL method.3、In vivo, the model of bearing human laryngocarcinoma in nude mice wasestablished by using human laryngeal squamous carcinoma cell line (Hep-2). The effect of TRAIL in inhibiting the LSCC was observed after it was administered to the animals. The ultramicrostructural changes of carcinoma were studied under microscope and transmission electron microscope during the experiment.ResultsThe results of our study showed that:1、The TRAIL and TRAIL-receptor expressed both in tissues of LSCC andlaryngeal normal tissues in the experiment of the RT-PCR and immunohistochemistry. The expression of the TRAIL, DcR1 and DcR2 in LSCC was weakly but highly in laryngeal normal tissues(P<0.01. The DR4, DR5 expressed in all tissues, there was no statistical difference in both groups (P>0.05) .2、The expression of TRAIL and DcR was higher in I and II stages, and lower in III and IV stages (P<0.05) . It was not statistically different of the expression of DR between the groups (P>0.05) . The TRAIL and DcR was expressed higher in well-differentiated LSCC, but weaker in poorly-differentiated LSCC (P<0.01), the expression of DR was not significant different in both groups (P>0.05) . All of these results were archived from the experiment of RT-PCR.3、The TRAIL and DcR was highly expressed in I and II stages but weakly in III and IV stages (TRAIL P<0.05, DcR P<0.01) . The expression of DR was not statistical different between the two groups (P>0.05) , and the TRAIL and DcR was highly expressed in well-differentiated LSCC, but weakly in poor-differentiated LSCC (P<0.01), there were no significant difference of the expression of DR detected between the two groups (P>0.05) .All of these results were obtained from the immunohistochemistry study.4、Under the inverted phase contrast microscope, we discovered that the higher concentration of TRAIL was obvious different from that of low concentration. After laryngeal squamous carcinoma cell Hep-2 was treated with TRAIL, there were some changes of the carcinoma cells, including nuclear fragmentation, the disappearance of chromatospherite, the chromatin pyknosis, the increased electronic density, the chondriosome swollen and the apoptotic body detected under electron microscope.5、MTT: In vitro, all different concentrations of TRAIL inhibited laryngeal squamous carcinoma cell’s growth. The inhibited growth ratio showed significant concentration-dependence. The concentrations for inducing apoptosis-ratio (TRAIL 1, 10, 100ng/ml) determined by flow cytometry was 11.49±0.36%, 22.31±0.82%, 59.64±1.10% respectively in the study group, and 3.13±0.12% in the control group, which was significantly different between these two groups(P<0.01).6、TUNEL: After the treatment with TRAIL, apoptosis occurred in the laryngeal squamous carcinoma cell Hep-2. The concentration for inducing apoptosis (TRAIL 1, 10, 100ng/ml) was 16.66±0.80%, 42.01±2.58%, 68.93±2.12%, and 3.32±0.19% in control group (P<0.01).7、All the experiments were successfully carried on in all the animal models of nude mice. The tumor of PBS group kept on growing and the volumes of the subcutaneous tumor were increasing. The condition of nude mice was dyscrasia. In contrast to the animals were given TRAIL (100ng/ml) , tumor grew slowly and only 1 nude mice died in the in treatment group, but 8 in the control group. The difference was significant in the average body weight and volume of the tumor tissue between the treatment group and the control group (P<0.01). And the tumor restrained percentage of the study group was 53.8%.8、The necrosis and apoptosis of the tumor cell were found under microscope and electron microscope in the control group. But, however, the growth of cells, nuclear fragmentation, the disappearance of chromatospherite and chromatin pyknosis was inhibited. And also the electron density increased and the apoptotic body detected.Conclusion1、The TRAIL and TRAIL-receptors are essentially expressed in LSCC andlaryngeal normal tissues. But expression degree and extent are different in different tissues. The expression of TRAIL and DcR has significant difference.2、In LSCC tissues, the TRIAL produces a marked effect in combining with itsreceptor, and eventually induces the apoptosis. In the normal laryngeal tissues, the decoy receptors competitively combine with the TRAIL, and thus inhibits the apoptosis.3、The level of TRAIL and DcR related with different clinical staging and different pathological stage.4、Laryngeal squamous carcinoma cell Hep-2 is sensitive to TRAIL. In vitro,TRAIL inhibits the growth of cells.The TRAIL shows a marked effect on inducing the apoptosis.5、The induced apoptosis of TRAIL shows significant concentration-independence.6、In vivo, TRAIL inhibits the growth of laryngeal tumor trough inducing apoptosis.更多还原