【作者】 莫珩；

【导师】 高文信；

【作者基本信息】 吉林大学 ， 口腔临床医学， 2004， 硕士

【摘要】 近年来，细胞凋亡与肿瘤的关系受到日益重视，认为多细胞生物通过细胞增殖与凋亡来维持自身稳定，若二者失衡，则可导致肿瘤发生。已有证据表明，许多肿瘤细胞具有抗凋亡作用，但其机制一直没搞清楚。核转录因子NF-κB (nuclear factor κB)是由Ranjan Sen等人于1986年首先在B 淋巴细胞中发现，得名于它能够与B细胞免疫球蛋白κ轻链基因的增强子κB序列特异地结合，是近年来发现的最重要的转录因子之一。众多的学者对NF-κB进行了广泛的研究，Science, Nature, Cell 等著名杂志登载了大量有关NF-κB研究的文章。近年研究表明，NF-κB在肿瘤的抗凋亡机制中起着关键性的作用。众所周知，肿瘤细胞的生物学特征是异常增殖和永生化，常常表现为凋亡不足。已发现，NF-κB的活化是肿瘤细胞对肿瘤坏死因子（Tumor Necrosis Factor,TNF）介导的凋亡产生抵抗性的主要机制。最近研究发现 NF-κB与几种人类恶性肿瘤，包括口腔鳞状细胞癌的发生、发展关系密切。本研究通过建立金地鼠颊囊癌变的动物模型，利用Western印迹和免疫组化技术检测NF-κB家族成员中的一个重要亚基p65和其抑制蛋白IκBα在金地鼠颊囊鳞癌发生、发展过程中的表达变化，并探讨其在金地鼠颊囊癌变过程中的作用及在口腔癌的早期诊断和基<WP=35>因治疗中的意义。迄今为止，已证明在哺乳类细胞中有五种NF-κB家族成员，其中p65（RelA）是NF-κB家族中的一个重要亚基，p65与p50形成的异二聚体是最常见的形式。该家族成员的N端均含有一段大约300多个氨基酸残基组成的高度保守的Rel同源域（Rel-homology domain,RHD）,其中包含DNA识别序列和核定位信号(nuclear localization signal,NLS)。在静息状态下，NF-κB异二聚体与其抑制蛋白IκB结合形成复合体存在于细胞浆中。通过非共价结合，IκB掩盖NF-κB的核定位信号而阻止NF-κB的核转位。一旦受到信号分子的刺激，IκBα被磷酸化后降解，NF-κB就从IκB中释放出来，激活并易位入核并与DNA NF-κB元件联结来诱导其依赖的基因转录。IκB是一类NF-κB的抑制蛋白。IκBα是IκB家族中发现最早和研究最清楚的成员，相对分子量为3.7KD,结构上由3部分组成：N端结构域可以在信号刺激下发生磷酸化，是其调控NF-κB活性的结构基础；中间是5个锚蛋白重复序列，为与NF-κB的结合部分；C端是一个四肽结构域，参与蛋白的基础转换。IκBα本身也是一个NF-κB的反应基因，在它基因启动子上游的调节区内具有κB位点，所以NF-κB的活化可上调IκBα，然后再通过这种反馈方式阻止NF-κB的活化过程，防止其过度活化。 为了探讨其在口腔粘膜上皮癌变过程中的表达情况，我们<WP=36>建立了采用DMBA诱导的金地鼠颊囊鳞癌的动物模型，并用组织病理学方法验证了动物实验的结果。从而系统观察了从正常颊粘膜、上皮单纯增生、上皮异常增生，最终演变成鳞癌过程中p65和IκBα的表达情况。Western blot法检测发现，在正常颊粘膜和上皮单纯增生组织中未见明显的p65表达，随着上皮异常增生的出现，p65表达增强（P ＜0.01），在鳞癌组织中，p65的表达进一步增强（P ＜0.01）。结果提示， p65可能在金地鼠颊囊鳞癌发生、发展过程中被激活。免疫组化研究表明，IκBα普遍表达于金地鼠颊粘膜的正常上皮和单纯增生上皮中，但多局限于基底细胞层和附近细胞的胞浆中。随着上皮异常增生的出现及异常增生程度的加重，IκBα表达水平降低（P ＜0.05）。根据文献资料及本研究的实验结果表明，在金地鼠颊囊癌变过程中IκBα被磷酸化而降解，从而释放了NF-κB，使之活化并进入细胞核中调节相关基因的转录。而当鳞状细胞癌发生后，IκBα的阳性表达明显增强（P ＜0.01），呈广泛强阳性表达。这可能是由于IκBα基因的启动子上有多个NF-κB结合序列，激活的NF-κB启动编码IκBα的基因转录，反馈性生成高表达量的IκBα，使NF-κB未再继续上升，从而两者在高水平上维持一种相互制约的关系。结果提示，IκBα在金地鼠颊囊鳞癌的发生发展过程中呈现一种动态变化过程。以上研究结果说明，p65、IκBα在金地鼠颊囊癌变过程中的表达异常，尤其是上皮异常增生阶段p65的表达水平上调而IκB<WP=37>α的表达下调可能是口腔粘膜上皮癌变过程中的早期事件，且可将其作为口腔早期癌变监测的生物学指标之一。同时，本研究结果也为通过核转录因子-κB的抑制剂IκBα基因转染而进行口腔鳞癌的基因治疗提供了分子生物学基础。更多还原

【Abstract】 The relation between apoptosis and tumor absorbed more eyes these years. It was believed that multicellular organism maintained their stability by cell proliferation and apoptosis, and tumor would occur once the balance was broken. Even through evidences showed that many tumor cells could resist apoptosis, the mechanism was still a mystery. Nuclear transcription factor κB (NF-κB) was firstly found by Ranjan Sen etc. in the B lymphocyte in 1986, which was one of the most important transcription factors and named from the special binding with the enhancer κB sequence of immunoglobulin kappa light chain gene. Comprehensive study on NF-κB was made by many researchers, and articles relating to NF-κB were often found in famous journals such as Science, Nature, Cell and so on. Recent founding showed that NF-κB played a key role in tumor anti-apoptosis mechanism. As we all know, the biological features of tumor cell were abnormal proliferation and immortalization, which usually expressed as apoptosis deficiency. The activation of NF-κB was the primary mechanism of tumor cell resisting the apoptosis mediated by tumor necrosis factor (TNF). New research showed that NF-κB had a close connection with the <WP=39>occurrence and develop of few human malignant tumors including oral squamous cell carcinoma (SCC).The study would establish the animal models of malignant transformation in hamster buccal mucosa by DMBA (0.5%), examine the expression and significance of p65——an important subunit of nuclear transcription factor κB (NF-κB) and its inhibitory protein IκBα in malignant transformation of hamster buccal mucosa., and discuss the mechanism of p65 processing in the malignant transformation of hamster buccal mucosa and early diagnosis and gene therapy of oral carcinoma.So far it was proved that there were 5 NF-κB family members in the mammalian cells, in which p65（RelA）was an important subunit, and the heterodimers formatted by p65 and p50 was the most common pattern. The N ends of this family members had a very conservative Rel-homology domain（RHD）combined by about 300 amino acid residues, including DNA recognition sequence and nuclear localization signal (NLS). The NF-κB heterodimers and its inhibitory protein IκBα formatted as complex in the cytoplasmic plasm when resting. By non-covalence combination, IκB hided the nucleic localization signal of NF-κB to prevent the nucleic dislocation of NF-κB. Once activated by the signal molecule, IκBα degraded after being phosphorylated, and <WP=40>NF-κB was released from IκBα, activated, translocated into the nuclear, and connected with DNA NF-κB component to induce the gene transcription which relied on.IκB was one kind of inhibitory protein of NF-κB. IκBα was a member of IκB family found earlist and studied most clear. The relative molecular weight of it was 3.7KD, and IκBα was combined with 3 parts in construction including: the base of controlling the activation of NF-κB, i.e. the N end structure domain which could start phosphorylation under the stimulation of signal; the connection part with NF-κB, i.e. 5 ankyrin repeated sequences; the C end, i.e. a tetra-peptide structure domain which took part in the basic transition of protein. IκBα itself was a react gene of NF-κB, and in the controlling domain located in the upper stream of gene promoter there was a κB site. Hence the activation of NF-κB could up-regulate IκBα, then inhibited the activating process of NF-κB by such a feedback manner to prevent the super-activation of it.To discuss the expression of p65 in the carcinomatous process of oral mucosa epithelia, we established the animal models of malignant transformation in hamster buccal mucosa by DMBA (0.5%), and verified the results of animal experiment with histophthology manner. The expression of p65and IκBα in the <WP=41>process covering normal buccal mucosa, epithelial hyperplasia, epithelial dysplasia and squamous cell carcinoma (SCC) were systemically examined. Western blot found that in normal buccal epit更多还原