【作者】 黄榕芳；

【导师】 余英豪；

【作者基本信息】 福建医科大学 ， 病理学与病理生理学， 2008， 硕士

【摘要】 【目的】通过腹腔注射DNA烷化剂N-甲基-N-亚硝基脲(MNU)诱导小鼠胸腺淋巴瘤,建立相应动物模型;应用组织形态学、免疫表型、超微结构观察及巢式聚合酶链反应(PCR)方法鉴定肿瘤细胞来源并分型。【方法】1.第一部分,选用6～8周龄C57BL/6小鼠52只,随机分实验组40只和对照组12只。实验组小鼠腹膜腔内注射新鲜配制MNU诱导液(50mg/kg体重);对照组腹膜腔内注射等量生理盐水,共注射2次(分别于第0、8周)。注射后观察动物一般情况,对濒死及死亡小鼠进行解剖观察。第22周采用颈椎脱位法处死全部小鼠,解剖检查胸腺淋巴瘤的发生及其他脏器情况。2.第二部分,应用光镜和透射电镜,对诱发的胸腺淋巴瘤及其侵袭脏器进行研究,分析其组织学及超微结构特点。通过免疫组织化学方法(所选抗体有CD3、CD20、TdT及PCNA)鉴定肿瘤细胞来源、分型。3.第三部分,采集8例实验组胸腺淋巴瘤组织及4例对照组胸腺组织,迅速放入液氮罐内,后置入-70℃冰箱冻存。提取基因组DNA,采用巢式PCR方法,对采集样本进行T细胞受体β链(TCRβ)和γ链(TCRγ)克隆性基因重排分析,并对TCRγ基因重排的PCR产物直接测序。【结果】1.实验后期,实验组小鼠体重明显下降。第22周,67.5%(27?40)实验组小鼠产生胸腺淋巴瘤。不同性别对成瘤率影响无明显差异。77.8%胸腺淋巴瘤合并其他脏器侵犯。2. 27例胸腺淋巴瘤光镜下主要表现为:胸腺结构破坏,代之以弥漫分布的中等大小淋巴样细胞,细胞形态比较一致,胞质稀少,核圆形、卵圆形或扭曲核,染色质细腻,核仁显著,核分裂象多见,可见“星空”现象。免疫组化显示瘤细胞表达CD3和TdT。超微结构观察,瘤细胞平均直径7.02±1.12μm,表面无突起,核形相对规则或伴有扭曲核,核仁靠近核膜,核浆比例高,胞质内细胞器极少,凋亡细胞易见。3. 8例胸腺淋巴瘤检测TCRβ和TCRγ均呈克隆性基因重排,阳性率100%;4例对照组胸腺组织均未检测到TCRβ或TCRγ克隆性基因重排。DNA序列测定证实TCRγ基因PCR扩增产物为基因重排产物。【结论】1.腹腔分次注射MNU可以诱导小鼠产生胸腺淋巴瘤,其生物学行为与人类肿瘤相似。2.结合光镜、免疫组化和电镜检查结果,证实所有肿瘤为胸腺T淋巴细胞起源的前驱淋巴母细胞淋巴瘤。3.巢式PCR TCRβ和TCRγ的基因重排检测及DNA序列分析证实,MNU诱导的小鼠胸腺淋巴瘤实际上是T淋巴细胞的单克隆性增生,是来源于T细胞的肿瘤。总之,MNU诱导的小鼠胸腺淋巴瘤在生物学行为、形态学、免疫表型和遗传学方面均与人类相应肿瘤极其相似,可作为人类这一肿瘤实体的理想动物模型。更多还原

【Abstract】 【Objective】To establish an animal model for thymic lymphoma in inbred mice induced by intraperitoneal injection of DNA alkylating agent N-methyl-N-nitrosourea (MNU), which would mimic this kind of human malignant lymphoma. Histopathologic, immunohistochemical, ultrastructural and nested PCR studies were performed to detect the the origin of tumor cells and to identify the subtype classification of induced tumors.【Methods】1. In the first part of our study, 52 male and female mice of the C57BL/6 strain at 6-8 weeks of age were randomly assigned to exposure and control groups, 40 and 12 animals respectively. Briefly, the exposure group were injected by the intraperitoneal route with MNU solution shortly after dissolution at a dosage of 50 mg/kg body weight. The control group received the same dosage of sterile 0.9% NaCl solution. The experimental animals were boosted 8 weeks later. Following injection of MNU, the mice were examined frequently. Seriously ill animals were killed and with mice found dead were completely autopsied. All mice were sacrificed by cervical dislocation for autopsy before the 22th experimental week. Complete gross examination was performed for detection of tumor masses.2. In the second part, thymic lymphomas as well as involved organs were fixed and routinely processed for light and electron microscopy examination to analyze histopathologic and ultrastructure features of the tumors. Sections were submitted to immunohistochemical staining with CD3, CD20, TdT and PCNA to identify the origin and subtype classification of the neoplasias.3. In the third part, 8 samples of thymic lymphoma and 4 samples of thymus tissue were frozen immediately in liquid nitrogen after removed and stored at -70℃. Genomic DNA was isolated from the thymic tumors and thymus tissues. Analysis with nested polymerase chain reaction(PCR) for TCRβand TCRγclonal gene rearrangement was carried out to determine the lineage and clonality of the T-cell lymphoma cells. The PCR products of TCRγgene rearrangement were purified and directly sequenced.【Results】1. The exposure group presented a mean body weight significantly lower than the control at the end of the experiment. At the 22th week, the proportion of MNU-treated animals developing thymic lymphoma after injection of MNU was 67.5%(27?40). No significant sex difference in the incidence was observed. Extensive invasion into other organs was found in 77. 8% of affected animal.2. Histopathologic study revealed that 27 cases of thymus became totally replaced by sheets of cells of the lymphoid series. The thymic tumors were densely infiltrated with relatively uniform, mitotically active, medium-sized cells with round to ovoid and sometimes convoluted nuclei that were surrounded by small amounts of cytoplasm. The chromatin pattern was finely granular and prominent nucleoli were present in most of the cells which had a high mitotic rate. There may be a“starry sky”pattern in some cases. All the tumors coexpressed CD3 and TdT. On transmission electron microscopic study, the mean diameter of the malignant lymphocytes was 7.02±1.12μm. Their nuclearcytoplasmic ratio was high, and their plasma membranes were smooth. The nuclear profiles were usually regular, with varying percentage of convoluted nuclei. Most nuclei contained one or two, usually eccentrically located nucleoli. Few cell organoids were observed in cytoplasm. Howere, apoptosis was common. 3. 8 cases of thymic lymohomas were all detected TCRβand TCRγclonal gene rearrangement. The positive rate was 100%. In the 4 cases of control group, no positive detected. DNA sequence analysis confirmed that the PCR products generated were indeed TCRγrearrangements.【Conclusions】1. As was confirmed, thymic lymohoma in mice can be induced by twice intraperitoneal administration of MNU. The biological behavior of the MNU-induced tumors resembled those of human thymic lymphomas derived from thymic T-cells.2. All the cases classified by histopathologic, immunohistochemical and ultrastructural studies as precursor lymphoblastic lymphoma were unquestionably related to the thymus origin and T-cell lineage.3. Just as determined by TCRβand TCRγgene rearrangement analysis and DNA sequence analysis, the MNU-induced thymic lymphoma in mice was of a T-cell origin and of monoclonal origin.In conclusion, MNU-induced thymic lymphomas in mice show strong parallels, all in their biological behavior and morphology and immunophenotype and genetics, to human thymic lymphoma. It confirmed that our cases may represent the murine counterpart of human tumor entity.更多还原