小鼠胸腺T细胞淋巴瘤模型的建立

发布时间：2018-04-24 14:00

  本文选题：恶性淋巴瘤 + T细胞淋巴瘤　； 参考：《福建医科大学》2008年硕士论文

【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。
[Abstract]:[purpose] Mouse thymic lymphoma was induced by intraperitoneal injection of DNA alkylator N-methyl-N-nitroso (MNUN), and the corresponding animal model was established by using histomorphology and immunophenotype. Ultrastructural observation and nested polymerase chain reaction (PCR) were used to identify the origin and type of tumor cells. [methods] 1. In the first part, 52 C57BL/6 mice aged 6 and 8 weeks were randomly divided into experimental group (n = 40) and control group (n = 12). The mice in the experimental group were injected with 50 mg / kg body weight of fresh MNU inducer in peritoneal cavity and the control group were injected with the same amount of normal saline twice (at the 0th week of 8 weeks respectively). The animals were observed after injection, and the near-death and dead mice were dissected and observed. All the mice were killed by cervical dislocation at the 22nd week. The occurrence of thymic lymphoma and other organs were dissected. 2. In the second part, the histological and ultrastructural characteristics of thymic lymphoma and its invasive organs were studied with light microscope and transmission electron microscope. The origin and typing of tumor cells were identified by immunohistochemical method (CD3, CD20, TdT and PCNA). 3. In the third part, 8 cases of thymic lymphoma in experimental group and 4 cases of thymic tissue in control group were collected and put into liquid nitrogen tank quickly, then frozen in -70 鈩，

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