【作者】 张永清；

【导师】 黄高昇；

【作者基本信息】 第四军医大学 ， 病理学与病理生理学， 2002， 博士

【摘要】 我国是淋巴瘤的高发地区之一，其中非霍奇金淋巴瘤(NHL)占淋巴瘤总数的90％，是我国最常见的淋巴造血系统恶性肿瘤之一。NHL的发病机制及同一组织类型的NHL在临床中表现出预后差异悬殊的原因一直悬而未决。随着被认为能够决定淋巴细胞最终命运的原癌基因BCL-6(B cell lympoma-6)的发现，给最终破解NHL的发病机制增加了希望。因为在非NHL中BCL-6基因调控区容易发生改变，其改变的频率可高达50％—87.5％。于是有人认为，相当一部分NHL的发生可能因为BCL-6基因调控区改变导致其表达产物BCL-6蛋白表达失调，表达失调的BCL-6又可致淋巴细胞正常分化受阻，最终导致淋巴瘤的发生。但目前仍有许多问题未能解决，如BCL-6是否同样表达于其它正常组织和肿瘤组织中?在NHL中BCL-6的表达水平是否与其基因调控区变化有必然联系?为了解决以上问题，我们进行了以下研究： 用常规免疫组织化学方法对人的淋巴组织、肝、肠、乳腺等10余种肿瘤组织及相应瘤旁组织中BCL-6表达情况进行了研究，染色阴性的标本用敏感性更高的免疫组织化学催化信号扩增法(CSA)进行验证。结果显示BCL-6特异地表达于反应性增生淋巴组织(RLH)的生发中心细胞及生发中心细胞起源的滤泡性淋巴瘤(FL)和86％弥漫性大B细胞淋巴瘤(DLBCL)。但BCL-6表达水平有显著区别，100％(17／17)RLH和77％(10／13) 第口旱民大学协士学位枉文·＋义纫昙 FL为中等阳性强度表达，23％（3／13）FL和 72％（ZI／29）DLBCL为强阳性表 达，肝、肠、乳腺等肿瘤组织和肿瘤旁组织以及小淋巴细胞淋巴瘤（SLL）、 套细胞淋巴瘤（MCL）、经典型霍奇金淋巳瘤均未见表达。通过PCR、克隆、 测序方法比较不同水平表达BCL－6 FL调控区序列改变倩况发现，强表达 BCL－6的FL其基因调控区均有不同程度突变，而中等强度表达的FL调控 区未见突变发生。BCL－6特异地表达于正常淋巴组织生发中心细胞，说明 BCL－6在生发中心形成过程中发挥了重要作用。对基因调控区变化分析结 果说明部分FL强表达BCL－6可能因其基因调控区变化所致，而强表达 BCL－6又可能与这部分FL的发生密切相关。 研究表明，BCL－6不仅与淋巴瘤的发生密切相关，在某些白血病细胞 分化时，其表达水平上调’川，还与其它多种肿瘤细胞的分化、凋亡密切相 关，甚至多数情况下，BCL－6单基因激活即可导致肿瘤细胞的分化或凋亡 ””皿知。而失分化和凋亡障碍正是白血病细胞的共性，研究如何诱导白血 病细胞分化和凋亡以及探讨诱导其分化和凋亡过程中发挥重要作用的关 键基因正是目前攻克白血病的主要突破口’““。为此，我们研究了BCL－6 在诱导白血病细胞分化和凋亡中的作用。主要研究内容及结果如下： l．分别以 HMBA（Hex。ethylene Bisacetamlde）、TPA（0－Tetra decanoy lphofbOI 13－aCBtsts）和h（hydTOXyeres）为诱导剂复策 K562细胞向单核 巨噬系、红细胞系及巨核细胞系分化的模型。在对模型进行形态学、细胞化 学及免疫组织化学的鉴定后，用 Northern Blot和 Western Blot方法检测了 对照组及向不同细胞系分化的K562细胞BCL－6表达情况。发现诱导前K562 细胞BCL－6 InRNA表达阴性，而在诱导其向不同细胞系分化时，K562细胞BCL－6 InRNA均有不同程度表达上调，但其向巨核细胞分化时BCL－6 InRNA表达水平 最高，分别是向单核细胞分化的1．5倍和向红细胞分化的3倍，而BCL－6蛋 白只在K562细胞向巨核细胞分化时表达阳性。用BCL－6反义寡核昔酸可有效 阻滞BCL－6表达及TPA诱导的K562向巨核细胞分化，说明BCL－6在TPA诱导 的K562细胞向巨核细胞分化过程中起到了重要作用。 2．用从传统中药中纯化的苦参碱诱导K562细胞，制备了K562细胞的 凋亡模型。在对这一模型进行系统鉴定后，用 Northern Blot和 Western Blot 方法检测发现，苦参碱可诱导K562细胞BCL－6在InRNA及蛋白质水平上表达 －3－ 剪口旱巨大学俗士学应沦文·＋丈匐昙 上调，采用反义技术作为“基因封条”特异封闭BCL－6蛋白表达时，苦参碱 诱导K562细胞的调亡细胞数也随之下降，说明BCL－6参与了苦参碱诱导K562 细胞凋亡的作用。 3．为进一步研究苦参碱诱导K562细胞凋亡的内部机制及BCLAs在这一 过程中的作用和地更多还原

【Abstract】 China is among the countries afflicted by high incidence of lymphomas. Of all the malignant lymphomas, non-Hodgkin’s lymphoma (NHL) makes up 90 percent. However, its pathogenesis and the significantly different clinical prognosis of the same kind of NHL remain to be revealed. With the discovery of BCL-6 proto-oncogene, which as is reported, regulates lymphocyte cell fate decisions, the discovery of the pathogenesis of lymphoma is almost with our reach. Because the 5’noncoding regulatory region of BCL-6 gene is found susceptible to translocation, mutation and deletion, the change frequency was up to 87.5% in NHL. Based upon this, some researchers think that the change in the regulatory region of BCL-6 will lead to the deregulation of expression of BCL-6 protein, and hi turn, the deregulated BCL-6 might result in lymphoma. However, there are still many questions to be answered, such as whether BCL-6 gene product is expressed widely in different human normal tissues and their neoplastic counterparts as well as whether the expression level of BCL-6 must be related to changes of regulatory region of BCL-6 gene. The present study was aimed at finding answers to the above questions.In the present study, expression of BCL-6 was studied in over 10 types of human normal tissues such as lymphoid node, liver, breast, intestine, etc. andtheir neoplastic counterparts with normal immunohistochemistry. Tissues with negative result were further tested with catalyzed signal amplification (CSA) for immunohistochemistry. The result indicates that expression of BCL-6 was localized specifically in the nuclei of cells of germinal center of all reactive hyperplasia lymphoid tissue (RHL), all the follicular lymphoma (FL) and 86 percent of 29 cases of diffuse large B-cell lymphoma (DLBCL). Nevertheless, BCL-6 expression levels in different tissues differed significantly . 100 percent of 17 RLH and 77% of 13 FL cases were positive with moderate expression; 23 percent of the rest of FL cases and 72 percent of 24 positive DLBCL cases were positive with intensive expression; and BCL-6 expression was negative in all other tissues. The difference in hypermutation of the 5’ noncoding region of BCL-6 gene between FLs with BCL-6 moderate expression and FLs with BCL-6 intensive expression was analyzed by using PCR, cloning and sequencing, mutations of the 5 non coding region of BCL-6 gene were found hi all 2 cases of FLs with BCL-6 intensive expression, and the changes were not found in FLs with BCL-6 moderate expression. The specific expression of BCL-6 in germinal center cells of normal lymphoid tissue may indicate the formation of the germinal center; and the mutation of regulatory region of BCL-6 gene may be the cause of intensive BCL-6 expression in a considerably small number of FLs; the intensive BCL-6 expression may be related to the pathogenesis of the relatively few FLs.Many researchers have reported that BCL-6 is closely related not only to pathogenesis of lymphoma but also to cell differentiation and apoptosis of many kinds of tumors. Expression of BCL-6 was increased when some kinds of leukemia cells were induced to differentiate. In most cases, activation of the single gene of BCL-6 induces cell differentiation or apoptosis, whereas the loss of differentiation and the apoptosis obstruction are the common characteristics of different leukemia cells. The study on how to induce leukemia cell differentiation and apoptosis and induce leukemia cells to differentiation or to apoptosis as well as the study on how to find the keygenes that respond to the differentiation and apoptosis of the induced leukemia cells are critical to a breakthrough in the therapy of leukemia.To identify the effects of BCL-6 gene on differentiation and apoptosis of induced leukemia cells, we conducted some experiments as follows.1. Three differentiation models of leukemia cell line K562 were established. HMBA (Hexamethylene Bisacetamide), TPA (0-Tetradecanoyl phorbol 13-acetate) and Hu (hydroxyurea) were selected to induce K562 cells directi更多还原