A20/LV-mCD99L2-A20鼠淋巴瘤动物模型的构建与HL
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摘要
研究背景
淋巴瘤是原发于淋巴造血组织的免疫系统恶性肿瘤。随着AIDS、器官移植、肿瘤放化疗免疫抑制的应用等近年来发病率急剧升高。
与非霍奇金淋巴瘤截然不同,霍奇金淋巴瘤(Hodgkin lymphoma,HL)以其相当独特的病理特征而倍受人们关注,其恶性成分-H/RS(Hodgkin/Reed-Sternberg,H/RS)细胞只占肿瘤组织的极少部分(＜1%),其余是大量以淋巴细胞为主的背景细胞。H/RS细胞的发生、发展机制一直悬而未决,而由于缺乏能够体现HL特殊病理学特征的动物模型,H/RS细胞与背景细胞相互关系的研究受到限制。有报道H/RS细胞的发生与CD99基因表达缺失有关;本研究组前期将慢病毒ShRNA质粒转染内源性mCD99L2(mouse CD99 antigen-like 2)基因表达阳性的鼠B淋巴瘤细胞株A20以筛选低表达mCD99L2基因的LV-mCD99L2-A20克隆株时发现其中出现类似人HL的H/RS样细胞,目前需要鉴定LV-mCD99L2-A20克隆株在反复传代过程中能否始终保持低表达mCD99L2的特性、能否始终出现类似人HL的H/RS样细胞、LV-mCD99L2-A20克隆株在生物学特性和免疫表型上与A20细胞有何差异,继而需要探讨LV-mCD99L2-A20克隆株在动物体内的成瘤特性及能否在BALB/c小鼠体内诱导出类似人HL的背景细胞等问题。
目前常见的淋巴瘤模型多为用淋巴瘤细胞株或病人淋巴瘤组织种植于免疫缺陷动物体内而建立的移植性淋巴瘤模型,这些模型在研究淋巴瘤分子机制中发挥了重要作用,但淋巴瘤为免疫系统恶性肿瘤,其发生与机体免疫状况有着密切的关系,因此无论是T细胞缺陷的裸小鼠还是T、B细胞联合免疫缺陷的SCID小鼠,由于其免疫功能的缺乏,无法模拟具有免疫功能的人体内环境,难以运用此类模型去揭示淋巴瘤的发生与机体之间的相互作用及机制,更加难以运用于HL模型中背景细胞的诱导,因此在选用LV-mCD99L2-A20细胞接种动物之前,有必要对鼠B淋巴瘤细胞株A20在有免疫功能的BALB/c小鼠体内成瘤的方式进行探讨,比较不同造模方式的优劣,同时也可为B淋巴瘤相关研究提供适合的实验平台。
另外,HL背景细胞的产生不仅与机体整体的免疫状况有关,也与H/RS细胞所诱导的免疫微环境有关。报道显示可能有多种细胞因子参与了H/RS细胞在体内的生存、增殖和逃避凋亡的过程,从而营造了H/RS细胞适宜的生存环境。细胞因子与HL及H/RS细胞之间的关系如何、A20与LV-mCD99L2-A20细胞在细胞因子的表达方面有无差异、在体内成瘤组织中A20与LV-mCD99L2-A20分别对细胞因子的表达产生何种影响、LV-mCD99L2-A20体内外的细胞因子表达能否有助于HL动物模型的构建等一系列问题有待解决,因此在分别构建了A20/LV-mCD99L2-A20动物模型后本研究将从细胞因子表达的角度初步探讨A20/LV-mCD99L2-A20细胞模型和动物模型与HL之间的关系。
目的
1、鉴定低表达mCD99L2的LV-mCD99L2-A20细胞亚系,以探究mCD99L2基因在HL的H/RS细胞形成中的作用,并为后续模型动物的构建奠定基础。
2、鼠源性B淋巴瘤细胞株A20以不同方式接种同源BALB/c小鼠,构建有免疫功能的鼠B淋巴瘤动物模型,探讨不同造模方式的优劣,为本研究后续工作奠定基础,并为B淋巴瘤相关研究提供适合的实验平台。
3、将LV-mCD99L2-A20细胞亚系接种BALB/c小鼠,进行成瘤情况观察、病理形态学观察、分子生物学鉴定和免疫标记的检测,探讨成瘤特点、成瘤组织病变特征与A20动物模型之间的差异及与HL之间的关系。
4、借助生物信息学软件对HL与细胞因子相关性进行检索和分析。利用蛋白芯片初步检测A20/LV-mCD99L2-A20细胞和BALB/c成瘤组织中细胞因子的表达、及差异,寻找LV-mCD99L2-A20细胞模型、动物模型与构建类人HL动物模型之间关系的相关依据。
方法
1、LV-mCD99L2-A20细胞亚系的鉴定
对前期构建的慢病毒质粒稳定干扰的LV-mCD99L2-A20克隆株进行体外培养,倒置显微镜、HE染色观察LV-mCD99L2-A20细胞的形态变化并进行计数,随机选择多代细胞进行DNA-PCR检测ShRNA干扰载体的整合,RT-PCR及荧光定量PCR检测目的基因mCD99L2的表达水平,免疫荧光观察mCD30分子标记,MTT法观察细胞体外增殖能力,流式细胞仪检测细胞周期及细胞的免疫表型(CD3、CD4、CD8、CD19、CD20、CD30)。
2、五种方式构建有免疫功能A20鼠B淋巴瘤动物模型
将鼠源性B细胞淋巴瘤细胞株A20经皮下接种、尾静脉注射、脾脏注射、腹腔注射于同源BALB/c小鼠和A20接种裸鼠成瘤组织移植BALB/c小鼠,观察动物成瘤时间、成瘤率、皮下肿瘤生长速度、内脏成瘤播散部位;取动物脏器行石蜡包埋、制备病理切片、HE染色观察病理学形态;运用流式细胞仪检测其瘤组织细胞悬液中CD3、CD4、CD8、CD19、CD30的表达;流式细胞仪检测正常BALB/c小鼠、接种成瘤小鼠和未成瘤小鼠外周血中淋巴细胞亚群的比例。
3、LV-mCD99L2-A20鼠淋巴瘤动物模型的构建
将经过鉴定的低表达mCD99L2基因的LV-mCD99L2-A20细胞亚系经皮下接种、尾静脉注射和裸鼠成瘤组织块移植的方法接种BALB/c小鼠,观察动物成瘤情况;取动物脏器行石蜡包埋、病理切片、HE染色观察病理学形态;计数成瘤切片中H/RS样细胞;免疫荧光检测瘤组织mCD30分子标记;运用流式细胞仪检测其瘤组织细胞悬液中CD3、CD4、CD8、CD19、CD30的表达。肿瘤组织进行原代培养,DNA-PCR检测载体的整合,RT-PCR检测mCD99L2基因mRNA水平。流式细胞仪检测小鼠外周血和脾脏中淋巴细胞亚群的比例。
4、A20/LV-mCD99L2-A20体内外细胞因子表达蛋白芯片初步检测
运用生物信息学软件GeneClip对HL和62种细胞因子进行文献检索,构建网络关系图。运用小鼠细胞因子功能分类蛋白芯片(RayBiotech mouse cytokineAntibody Arrays)对A20细胞和LV-mCD99L2-A20细胞、A20细胞BALB/c皮下成瘤组织和LV-mCD99L2-A20细胞BALB/c鼠皮下成瘤组织进行蛋白质抽提、蛋白质定量,膜封闭和孵育、62种小鼠细胞因子检测、图像采集和数据分析,对差异细胞因子进行初步分析。
结果
1、LV-mCD99L2-A20细胞亚系的鉴定
(1)传代过程中倒置显微镜观察和HE染色均发现LV-mCD99L2-A20细胞中存在类似H/RS细胞的大细胞。随机抽取A、B、C、D代LV-mCD99L2-A20细胞计数其中的大细胞发现较A20细胞中的大细胞多,差异有显著性(P＜0.05)。
(2)抽提多代LV-mCD99L2-A20细胞DNA进行PCR均能检测出ShRNA干扰载体稳定整合至A20细胞基因组。
(3) RT-PCR及荧光定量PCR检测目的基因mCD99L2均较A20细胞呈低水平表达,干扰效率约为50%。
(4) MTT检测发现LV-mCD99L2-A20细胞体外增殖能力较对照A20组和空载体组减慢,差异有显著性(P＜0.05)。
(5)流式细胞仪检测细胞周期发现LV-mCD99L2-A20细胞S期比例与A20细胞无显著性差异(P＞0.05),但是G2期较A20细胞长,差异有显著性(P＜0.05)。
(6)免疫荧光检测LV-mCD99L2-A20细胞及其中的大细胞CD30呈阳性表达。
(7) LV-mCD99L2-A20细胞的CD3表达(7.47±1.27%)较A20细胞(18.93±3.87%)降低,CD8表达(8.33±3.89%)较A20细胞(18.27±3.45%)降低,CD20表达(13.10±5.16%)较A20细胞(35.33±2.25%)降低,CD30表达(76.0±12.44%)较对照A20细胞(41.70±2.60%)升高,差异有显著性(n=3,P＜0.05),而CD4、CD19差异不显著(P＞0.05)。空载体组与对照A20细胞CD抗原表达无显著差异(P＞0.05)。
2、五种方式构建有免疫功能A20鼠B淋巴瘤动物模型
(1)皮下成瘤:BALB/c小鼠皮下注射2×10~5组(n=7)未成瘤,2×10~6组(n=7)、2×10~7组(n=7)和裸鼠瘤组织移植BALB/c小鼠组(n=7)成瘤率皆为100%,成瘤时间分别为15.29±3.2天、7.0±0.82天、6.29±0.49天。
(2)内脏成瘤:BALB/c小鼠尾静脉注射2×10~6组、2×10~7组、脾脏注射组、腹腔注射组成瘤率分别为71.4%(5/7)、100%(7/7)、71.4%(5/7)、14.3%(1/7),成瘤时间分别为76.8±12.0天、26.1±7.99天、32.6±5.99天和27天。尾静脉成瘤部位播及肝脏、脾脏、胰腺、肾脏、食道、胃、肠、肠系膜、脑、淋巴结、骨、子宫、肌肉等多脏器和组织。
(3) BALB/c鼠A20成瘤组织镜下观察:瘤细胞呈弥漫性分布,大小一致,细胞核大、圆形或不规则形,核仁明显,胞质中等量、嗜碱性,类似人弥漫大B细胞淋巴瘤。
(4)流式细胞仪检测成瘤组织细胞悬液(n=8)CD3、CD4、CD8、CD19、CD30的表达阳性细胞比例分别为(%):49.27±23.75、6.07±3.65、51.2±23.1、67.06±16.39、37.93±17.03。
(5)流式细胞仪检测外周血淋巴细胞亚群:各组间CD3、CD4阳性细胞比例存在显著性差异。接种A20细胞成瘤BALB/c小鼠与正常BALB/c小鼠相比CD3(P=0.014)、CD4(P=0.009)阳性细胞比例下降,差异有显著性。接种A20细胞成瘤BALB/c小鼠与接种A20细胞未成瘤BALB/c小鼠相比CD3(P=0.004)、CD4(P=0.006)阳性细胞比例下降,差异显著。接种A20细胞未成瘤BALB/c小鼠与正常BALB/c鼠相比CD3(P=0.592)、CD4(P=0.867)、CD8(P=0.398)、CD19(P=0.510)阳性细胞比例和CD4/CD8(P=0.545)比值差异皆不显著。
3、LV-mCD99L2-A20鼠淋巴瘤动物模型的构建
(1) LV-mCD99L2-A20接种后成瘤情况:皮下接种2×10~7细胞于4只裸鼠(6个位点),100%成瘤,平均成瘤时间为13.33±4.63天,生长速度较A20裸鼠皮下接种2×10~7组生长缓慢。皮下注射2×10~7LV-mCD99L2-A20细胞于BALB/c小鼠14只,7.3%(1/14)成瘤;成瘤时间10天。裸鼠瘤组织移植BALB/c小鼠14只,7.3%(1/14)成瘤,成瘤时间6天。BALB/c小鼠尾静脉注射组28只,三月宰杀时均未成瘤。
(2)病理形态:裸鼠和BALB/c小鼠成瘤组织光镜观察瘤细胞大小不一,弥漫分布,可见散在的胞浆丰富的大细胞,呈双核、多核或不规则核,核仁大,极其类似人HL的H/RS细胞形态。BALB/c小鼠H/RS细胞周围存在一定量淋巴细胞的浸润;瘤组织流式检测CD3、CD4、CD8、CD19和CD30皆呈较高表达。
(3) BALB/c小鼠瘤组织原代培养:DNA-PCR检测LV-mCD99L2干扰载体整合在细胞基因组,RT-PCR显示mCD99L2基因mRNA水平较A20细胞为低。
(4)流式细胞仪检测外周血淋巴细胞亚群:各组间CD3、CD4、CD8、CD19阳性细胞比例存在显著差异。接种A20细胞未成瘤BALB/c小鼠与正常BALB/c小鼠各指标差异无显著性(P＞0.05)。接种LV-mCD99L2-A20细胞未成瘤BALB/c小鼠与正常BALB/c小鼠相比外周血CD3(P=0.014)、CD4(P=0.018)阳性细胞比例下降而CD19(P=0.012)阳性细胞比例升高,差异有显著性;而CD8(P=0.075)阳性细胞比例差异不显著。接种LV-mCD99L2-A20细胞未成瘤BALB/c小鼠与接种A20细胞未成瘤BALB/c小鼠相比CD3(P=0.004)、CD4(P=0.012)和CD8(P=0.007)阳性细胞比例下降,差异有显著性;而CD19(P=0.052)阳性细胞比例差异不显著。
(5)脾脏淋巴细胞亚群:接种A20细胞未成瘤BALB/c小鼠与正常BALB/c惺笙啾菴D3(P=0.003)、CD4(P=0.007)、CD8(P=0.000)阳性细胞比例下降,CD19(P=0.004)阳性细胞比例和CD4/CD8(P=0.000)比值升高,差异有显著性。接种LV-mCD99L2-A20细胞未成瘤BALB/c小鼠与正常BALB/c小鼠相比CD3(P=0.000)、CD4(P=0.001)、CD8(P=0.000)阳性细胞比例下降而CD19(P=0.000)阳性细胞比例和CD4/CD8(P=0.000)比值升高,差异有显著性。接种LV-mCD99L2-A20细胞未成瘤BALB/c小鼠与接种A20细胞未成瘤BALB/c小鼠相比CD3(P=0.009)、CD4(P=0.020)阳性细胞比例下降,CD19(P=0.020)阳性细胞比例升高,差异有显著性;CD8(P=0.170)阳性细胞比例和CD4/CD8(P=0.646)比值无显著性差异。
4、A20/LV-mCD99L2-A20细胞和组织细胞因子表达的蛋白芯片初步检测
(1) LV-mCD99L2-A20细胞较A20细胞上调(≥1.5倍)的细胞因子有CD30T、IL-12p40/p70、IL-3、IFNγ、CXCL16、MIP-1α、CD40,其中CD30T表达上调2.91倍。未见表达下调超过1.5倍的细胞因子表达。
(2)LV-mCD99L2-A20细胞BALB/c小鼠成瘤组织较LV-mCD99L2-A20细胞上调≥1.5倍的细胞因子主要有VCAM-1、MIP-1γ、MIG、IL-10、RANTES、PF-4、CXCL16、VEGF、MIP-3α、P-Selectin,其中VCAM-1、MIP-1γ、MIG、IL-10上调≥2倍;下调≥1.5倍的细胞因子主要有IL-6、Eotaxin、TNFα、TIMP-1、SCF、IFNγ、IL-3、CD30T、IL-5、IGFBP-6、TECK、L-Selectin、Fas Ligand、IL-3 Rb、IL-9,其中IL-6下调3倍以上。
(3)LV-mCD99L2-A20细胞接种BALB/c小鼠成瘤组织较A20细胞接种BALB/c小鼠成瘤组织中下调(≥1.5倍)的细胞因子如Eotaxin、IGFBP-6、IL-12 p40/p70、IGFBP-5、MIP-1α、KC、CXCL16,其中Eotaxin、IGFBP-6下调超过2倍;而上调(≥1.5倍)的细胞因子仅是VEGF。
结论
1、LV-mCD99L2-A20细胞亚系中存在类似人H/RS细胞形态的H/RS样细胞,LV-mCD99L2-A20细胞在增殖能力、细胞周期和免疫表型方面较类似人HL。
2、A20细胞BALB/c小鼠皮下移植瘤模型成瘤时间短、尾静脉接种式成的血行播散性模型成瘤部位广泛,均为研究有免疫功能鼠B淋巴瘤适宜的实验动物模型。
3、LV-mCD99L2-A20细胞较A20细胞在BALB/c鼠难以成瘤,但在成瘤组织中出现似人HL的H/RS样细胞,伴有一定程度的背景淋巴细胞的浸润。
4、LV-mCD99L2-A20细胞较A20细胞上调了一些与报道中的HL或H/RS细胞相关的细胞因子;LV-mCD99L2-A20细胞与A20细胞在BALB/c小鼠体内成瘤组织中细胞因子表达不同。
创新之处
本研究从体外实验、裸鼠成瘤实验和BALB/c鼠成瘤实验三个水平,从形态学、免疫表型、生物学行为三个主要方面并结合小鼠免疫功能初步检测和细胞因子表达蛋白芯片初步检测,较为系统地对比了A20细胞与LV-mCD99L2-A20细胞在细胞模型之间、动物模型之间的差异,揭示了mCD99L2基因低表达对体外、体内诱导H/RS样细胞具有重要作用;提示了LV-mCD99L2-A20 BALB/c鼠模型在出现H/RS样细胞的同时其良好的预后可能与HL具有某种相似性;结合体内外细胞因子表达的差异分析了LV-mCD99L2-A20细胞模型、动物模型与构建HL之间的相关性,为探索性的研究,为构建稳定的HL动物模型奠定了坚实的基础。
Backgroud
Lymphomas are immune system malignant neoplasms of cells native to lymphoid tissue.Along with the wide use of immunosuppressant for organ transplant, AIDS occurence,and chemotherapy for tumors,the occurrences of lymphomas have drastically increased in recent years.
Different from non-Hodgkin's lymphoma(NHL),Hodgkin's lymphoma(HL) is a peculiar form of malignancy in which the clonal B cell population,the so called Hodgkin/Reed-Sternberg(H/RS) cells and their variants,is responsible for less than 1%of the bulk of the tumor.The mechanism of development of HL and H/RS cell is still illusive.The relationship between H/RS cell and background cells is also restricted for lack of suitable animal model which mimics the characteristics of HL. There are reports that human H/RS cell is formed for the low expression of CD99 (MIC2).Our research work have previously transfected the mouse B lymphoma cell line A20,which expressed mouse CD99 antigen-like 2 gene,using Letivirus ShRNA vector and constructed subseries of A20 cell line with low mCD99L2 gene expression,named "LV-mCD99L2-A20".Among the LV-mCD99L2-A20 cells,giant cells like human HL H/RS cells were found.Whether these giant cells could consistently present during continuous passage culture of LV-mCD99L2-A20 cells and what are the immunophenotypes of these cells? How about the tumors induced by LV-mCD99L2-A20 cells in the nude mouse and BALB/c mouse? What's the difference between LV-mCD99L2-A20 cells and A20 cells in mouse with nomal immune system?These problems need to be investigated.
The animal model people often used were engrafted immunodeficiency animals inoculated with lymphoma cell lines or the tissue from lymphoma patients,which were of great importance during the research into molecular mechanisms of lmphomas.Most of them are immunodeficiency animals.Lymphoma is a kind of tumor envolved with body immune system.For lack of T lymphocyte or/and B lymphocyte,neither the nude mouse nor the severe combined immunodeficiency mouse could mimic the immune enviroment of human body and it is hard to use these models to reveal the interaction between lymphoma and immune system of human body.Therefore these animal models are limited in the research of clinic therpy and drug usage.
The abundant reactive cells in HL were related not only with whole immune system of the body,but the immuno-microenviroment induced by H/RS cells.It has been postulated that the presence of abundant reactive cells in HL tissue is due to the production of several kinds of cytokines and/or chemokines.Really what is the relationship between HL H/RS cell and cytokines? Is there any difference in the cytokines' expression between A20 cells and LV-mCD99L2-A20 cells? What kinds of cytokines will be induced in BALB/c mouse by A20 cells and LV-mCD99L2-A20 cells respectively? Will the secretion of cytokines in LV-mCD99L2-A20 cells and tumor tissues benefit the construction of real HL animal model? After the costruction of A20/LV-mCD99L2-A20 animal model,our work will discuss the relationship between LV-mCD99L2-A20 cell model and/or animal model and costruction of HL.
Objective
1.Identifying the subseries which express low level of mouse CD99 antigen-like 2 gene(mCD99L2) of mouse B lymphoma cell line(A20),named LV-mCD99L2-A20, so as to investigate the importance of mCD99L2 in the formation of Hodgkin/ Reed-Sternberg(H/RS) like cell.
2.Inoculating mouse B lymohoma cell line A20 to homologic BALB/c mouse in different ways,so as to construct mouse B lymphoma animal models and compare the difference between them,thus providing suitable animal models for research in B cell lymphoma.
3.Using LV-mCD99L2-A20 cells to inoculate BALB/c mouse,investigating the tumor formation,observing its pathological characteristics,identifying its molecular biology and detecting its immunophenotypes so as to compare the biological difference between in vivo and in vitro,comparing the pathological characteristics between LV-mCD99L2-A20 cell-induced tumors and A20 cell-induced tumors,thus analyzing the relationship between the difference and human HL.
4.Applying the software of bioinformatics to retrieve and analyze the relationship between HL and cytokines.Using mouse cytokine antibody arrays to investigate the expression of cytokine in the cells and tumor tissues,comparing its difference and analyzing the relationship between the diffenrence and construction of human HL like animal model,thus providing experimental data for our profound research work.
Method
1.During the period of continuous passage culture of A20 cells formerly transfected with LentiVirus-mCD99L2 vector,named LV-mCD99L2-A20 cells,choosing cells of different passages respectively:examining the integrated status of vector using DNA-PCR;analyzing RNA interference efficiency by RT-PCR and real time RT-PCR, observing morphological characteristics and counting numbers of giant cells under light microscope,detecting expression of mouse CD30 molecules in LV-mCD99L2 -A20 cells by immunofluorenscence,observing the growth curve by MTT and detecting the cell cycle and immunophonotypes of A20 cells and LV-mCD99L2-A20 cells such as CD3,CD4,CDS,CD19,CD20,CD30.
2.Innoculating mouse B lymohoma cell line A20 to homological BALB/c mouse by subcutaneous inoculation,intravenous injection,spleen inoculation,intraperitoneal injection and transplantation of tissue from nude mouse,observing the tumor formation,paraffin imbedding the organs of animals,pathological section and HE staining to observe its pathological characteristics.Detecting CD3,CD4,CD8,CD19, CD30 expression of the cell suspension of tumor tissues with Fluorescence Activated Cell Sorter(FACS) technology.Comparing the difference of lymphocyte subpopulation in normal mouse and mouse with or without tumors.
3.Using identified LV-mCD99L2-A20 cells to inoculate homologic BALB/c mouse by subcutaneous inoculation and intravenous injection,observing the tumor formation,paraffin imbedding the organs of animals,pathological section and HE staining to observe its pathological characteristics,detect the expression of CD3,CD4, CD8,CD19,CD30 in cell suspension of tumor tissues by FACS and also compare the difference of lymphocyte subpopulation in peripheral blood and spleen of normal mouse and mouse without tumors after A20 inoculation and LV-mCD99L2-A20 inoculation.
4.Using bioinformatics software Geneclip to retrieve the articles about the HL and/or H/RS cell and 62 kinds of cytokines,analyzing the relationship between them.Using RayBiotech mouse cytokine Antibody Arrays to investigate the expression of cytokines in A20 and LV-mCD99L2-A20 cells and their related tumor tissues, extracting and quantitating proteins,sealing and incubating membranes,detecting expressions of cytokines,imaging photos and analyzing the difference between them.
Result
1.Identification of subseries of A20 with low expression of mCD99L2 gene (LV-mCD99L2-A20):
(1) Giant cells like Hodgkin/Reed-Sternberg cells(H/RS-like cell) were found during continuous passage culture of LV-mCD99L2-A20 cells.The number of giant cells in A,B,C,D passage cells were more than those in A20 cell line,with dramatic difference(P＜0.05)
(2) During continuous passages of LV-mCD99L2-A20 cells,282 bp fragments of LV-mCD99L2 ShRNA vector were detected by DNA extraction and following PCR reaction.
(3) Expression of mCD99L2 gene mRNA in LV-mCD99L2-A20 cells was lower (about 50%)than those in A20 control group revealed by RT and real-time PCR.
(4) The proliferating ability of LV-mCD99L2-A20 cell deteced by wethod of MTT is slower than the contral A20 group and mock group,with dramatic difference (P＜0.05).
(5) Cell cycle detection by FACS revealed that S phase of each group is of no significant difference(P＞0.05),while the G2 phase is longer than the contral A20 group and mock group,with significent difference(P＜0.05).
(6) LV-mCD99L2-A20 cells,including giant cells,were positive labeled by mouse CD30 antibody in immunofluorenscence test.
(7) The CD3 expression of LV-mCD99L2-A20 cell(7.47±1.27%)is lower than A20 cell(18.93±3.87%),CD8 expression of LV-mCD99L2-A20 cell(8.33±3.89 %)is lower than A20 cell(18.27±3.45%),CD20 expression of LV-mCD99L2-A20 cell(13.10±5.16%)is also lower than A20 cell(35.33±2.25%),while CD30 expression of LV-mCD99L2-A20 cell(76.0±12.44%)is higher than A20 cell (41.70±2.60%),all with significent difference(n=3,P＜0.05).The CD4 and CD19 expression of LV-mCD99L2-A20 cell were of no significant difference with A20 cell (P＞0.05).The mock group is of no significant difference with A20 cell in each CD antigen ex[ression(P＞0.05).
2.Construction of animal model using mouse B lymphoma A2O cell line using different methods:
(1) There are no tumor formed in 2×10~5 BALB/c mouse group,while 100%tumor formation in the BALB/c mouse of subcutaneous inoculation(2×10~6 and 2×10~7) groups and tissue(from nude mouse) trans-plantation group after inoculating about 15.29±3.2 days,7.0±0.82 days and 6.29±0.49 days,respectively. (2) There are 71.4%(5/7) and 100%(7/7) tumor formation in intravenous injection (2×10~6 and 2×10~7) group respectively,71.4%(5/7) in spleen inoculation group and 14.3%(1/7) in intraperitoneal injection group after about 76.8±12.0 days,26.1±7.99 days,32.6±5.99 days and 27 day.The tumor formed in several organs of mouse,such as liver,spleen pancrease,kidney,esophagus,stomach,intestine,mesentery,brain, lymphocytic node,bone,uterus and muscles et al.
(3) The tumor cells diffused with large round or irregular nucleus in the tissues, nucleoli is obvious and cytoplasm is light staining,somewhat like diffuse large B cell lymphoma(DLBCL).
(4) The expression of CD3、CD4、CD8、CD19、CD30 in the cells from tumor tissues(%) are 49.27±23.75、6.07±3.65、51.2±23.1、67.06±16.39 and 37.93±17.03, respetively.
(5) FACS detection of proportions of lymphocyte subsets in peripheral blood show significant differences in CD3 and CD4 expressions between each groups.The proportions of lymphocyte subsets in the blood of mouse with tumor genesis after A20 cell inoculation are lower in CD3(P=0.014) and CD4(P=0.009) expression than those of normal BALB/c mouse and also lower in CD3(P=0.004) and CD4(P=0.006) expression than those in mouse without tumor genesis after A20 cell inoculation.The proportions of lymphocyte subsets in the blood of mouse without tumor genesis after A20 cell inoculation are of no significant difference from those of normal BALB/c mouse:CD3(P=0.592),CD4(P=0.867),CD8(P=0.398),CD19(P=0.510) and CD4/CD8 (P=0.545) ratio.
3.Construction of animal model using identified LV-mCD99L2-A20 sbseries:
(1) There are 100%tumor formed in the nude mouse of subcutaneous inoculation (2×10~7) group after average about 13.33±4.63 days,while only 7.1%(1/14) tumor formed in the BALB/c subcutaneous inoculation(2×10~7) group and nude tumor tissues transplanting group,after 10 days and 6 days,respectively.Intravenously inoculated BALB/c mouse group(n=28) has no tumor formed till three months after inoculation.
(2) Tumor cells in nude mouse and BALB/c mouse indicated different sizes under light microscope,diffuse contribution,large and deep stained nucleus,sparse binucleated,polynucleated observation,which was similar to pathological characteristics of human HL H/RS cells.Besides,lymphocytes infiltrated into tumor tissues of BALB/c mouses,with high expression of CD3+ T lymphocyte,CD4+ T lymphocyte,CD8+ T lymphocyte and high CD30 expression.
(3) During primary culture of tumor tissues,282 bp fragments of ShRNA vector were detected in LV-mCD99L2-A20 B1 cells.Expression of mCD99L2 gene mRNA in LV-mCD99L2-A20 B1 cells was lower than A20 control group revealed by RT-PCR.
(4) FACS detection of proportions of lymphocyte subsets in peripheral blood show significant differences in CD3,CD4,CD8 and CD19 expressions between each groups.The proportions of lymphocyte subsets in the blood of mouse without tumor genesis after LV- mCD99L2-A20 cells inoculation are of significant differences from those of normal BALB/c mouse--lower in CD3(P=0.014),CD4(P=0.018) and higher in CD19(P=0.012) expression,while are of no significant differences in CD8(P=0.075).The proportions of lymphocyte subsets in the blood of mouse without tumor genesis after LV- mCD99L2-A20 cells inoculation are of significant differences from those of A20 inoculated BALB/c mouse -- lower in CD3(P=0.004), CD4(P=0.012) and CD8(P=0.007) expression while no significant differences in CD19(P=0.052) expression.
(5) FACS detection of proportions of lymphocyte subsets in spleen show significant differences between each groups.The proportions of lymphocyte subsets in the spleen of mouse without tumor genesis after LV-mCD99L2-A20 cells inoculation are of significant differences from those of normal BALB/c mouse --lower in CD3 (P=0.000),CD4(P=0.001),CD8(P=0.000) expressions while higher in CD19(P=0.000) expressions and CD4/CD8(P=0.000) ratio.The proportions of lymphocyte subsets in the spleen of mouse without tumor genesis after LV-mCD99L2-A20 cells inoculation are of significant differences from those of A20 inoculated BALB/c mouse - lower in CD3(P=0.009),CD4(P=0.020) expressions,higher in CD19(P=0.020) expression, while no significant differences in CD8(P=0.170) and CD4/CD8(P=0.646) ratio.
4.Expression of cytokines in A20 / LV-mCD99L2-A20 cells and tissues:
(1) The up-regulated cytokines(≥1.5 folds) in LV-mCD99L2-A20 cells,when compared with A20 cells,include CD30T,IL-12p40/p70,IL-3,IFNγ,,CXCL16, MIP-1αand CD40,among which CD30T is up-regulated by 2.91 folds.There is no down-regulated(≥1.5 folds) cytokines in LV-mCD99L2-A20 cells compared with A20 cells.
(2) The up-regulated cytokines(≥1.5 folds) in LV-mCD99L2-A20 cell-induced tumor in BALB/c mouse,compared with those in LV-mCD99L2-A20 cells,include VCAM-1,MIP-1γ,MIG,IL-10,RANTES,PF-4,CXCL16,VEGF,MIP-3αand P-Selectin,among which VCAM-1,MIP-1γ,MIG and IL-10 up-regulated in excess of two folds.The down-regulated cytokines(≥1.5 folds) include IL-6、Eotaxin、TNFα、TIMP-1,SCF,IFNγ,IL-3,CD30 T,IL-5,IGFBP-6,TECK,L-Selectin,Fas Ligand,IL-3 Rb and IL-9,among which IL-6 down-regulated in excess of three folds.
(3) When compared with A20 cell-induced tumor,the down-regulated cytokines(≥1.5 folds) include Eotaxin,IGFBP-6,IL-12 p40/p70,IGFBP-5,MIP-1α,KC and CXCL16 in LV-mCD99L2-A20 cell-induced tumor in BALB/c mouse,among which Eotaxin and IGFBP-6 down-regulated in excess of two folds,while the up-regulated cytokines(≥1.5 folds) is only VEGF.
Conclusion
1.There exist giant cells with morphologic characteristics that like human HL H/RS cells in the subseries of A20 which show low expression of mCD99L2 gene(LV-mCD99L2 -A20 cell).The biological characteristics and immunophotypes of LV-mCD99L2-A20 cell much more mimics HL than A20 cell.
2.The subcutaneously transplanted tumors of BALB/c mouse are formed in a shorter time period after A20 cell inoculation than those in other animal models.The hematogenous disseminated animal model by more than 10~6 A20 cell inoculation is involved with several visceral organs.These animal models provide suitable animal models for research of B lymphomas in mouse with nomal immune function.
3.LV-mCD99L2-A20,when inoculated into BALB/c mouse,has lower tumor genesis compared with A20 inoculation,while more giant cells with the morphologic characteristics that like human HL H/RS cells dispersed in LV-mCD99L2-A20 cell-induced tumors than in A20-induced tumors in BALB/c mouse,accompanied by some infiltrating lymphocytes.
4.LVomCD99L2-A20 cell is more likely to express cytokines usually appeared in HL cell lines or in H/RS cells.There exist differences between the cytokine expressions of LV-mCD99L2-A20 cell-induced tumors and A20-induced tumors in BALB/c mouse.
New point
From three levels:in vitro investigation,nude mouse and BALB/c mouse in vivo investigation,the research compared the fifferences systematically between A20 and LV-mCD99L2-A20 cell models,between their animal models by morphological observation,detection of immunophonotypes and biological characteristics combining with primary investigation of immune function and cytokines expressions.The results revealed and confirmed that the low expression of mCD99L2 gene is of great importance to the construction of H/RS-like cell model in vitro and in vivo and also revealed a better prognosis of LV-mCD99L2-A20 cells than A20 cells in animal with normal immune function,which may be some similar to genesis of HL. Through primary detection of cytokines in vitro and in vivo,analyzing the relationship between LV-mCD99L2-A20 cell model,animal model and construction of HL animal model.All of these made a better basis for prolonged construction of HL animal model.
淋巴瘤是原发于淋巴造血组织的免疫系统恶性肿瘤。随着AIDS、器官移植、肿瘤放化疗免疫抑制的应用等近年来发病率急剧升高。
与非霍奇金淋巴瘤截然不同,霍奇金淋巴瘤(Hodgkin lymphoma,HL)以其相当独特的病理特征而倍受人们关注,其恶性成分-H/RS(Hodgkin/Reed-Sternberg,H/RS)细胞只占肿瘤组织的极少部分(＜1%),其余是大量以淋巴细胞为主的背景细胞。H/RS细胞的发生、发展机制一直悬而未决,而由于缺乏能够体现HL特殊病理学特征的动物模型,H/RS细胞与背景细胞相互关系的研究受到限制。有报道H/RS细胞的发生与CD99基因表达缺失有关;本研究组前期将慢病毒ShRNA质粒转染内源性mCD99L2(mouse CD99 antigen-like 2)基因表达阳性的鼠B淋巴瘤细胞株A20以筛选低表达mCD99L2基因的LV-mCD99L2-A20克隆株时发现其中出现类似人HL的H/RS样细胞,目前需要鉴定LV-mCD99L2-A20克隆株在反复传代过程中能否始终保持低表达mCD99L2的特性、能否始终出现类似人HL的H/RS样细胞、LV-mCD99L2-A20克隆株在生物学特性和免疫表型上与A20细胞有何差异,继而需要探讨LV-mCD99L2-A20克隆株在动物体内的成瘤特性及能否在BALB/c小鼠体内诱导出类似人HL的背景细胞等问题。
目前常见的淋巴瘤模型多为用淋巴瘤细胞株或病人淋巴瘤组织种植于免疫缺陷动物体内而建立的移植性淋巴瘤模型,这些模型在研究淋巴瘤分子机制中发挥了重要作用,但淋巴瘤为免疫系统恶性肿瘤,其发生与机体免疫状况有着密切的关系,因此无论是T细胞缺陷的裸小鼠还是T、B细胞联合免疫缺陷的SCID小鼠,由于其免疫功能的缺乏,无法模拟具有免疫功能的人体内环境,难以运用此类模型去揭示淋巴瘤的发生与机体之间的相互作用及机制,更加难以运用于HL模型中背景细胞的诱导,因此在选用LV-mCD99L2-A20细胞接种动物之前,有必要对鼠B淋巴瘤细胞株A20在有免疫功能的BALB/c小鼠体内成瘤的方式进行探讨,比较不同造模方式的优劣,同时也可为B淋巴瘤相关研究提供适合的实验平台。
另外,HL背景细胞的产生不仅与机体整体的免疫状况有关,也与H/RS细胞所诱导的免疫微环境有关。报道显示可能有多种细胞因子参与了H/RS细胞在体内的生存、增殖和逃避凋亡的过程,从而营造了H/RS细胞适宜的生存环境。细胞因子与HL及H/RS细胞之间的关系如何、A20与LV-mCD99L2-A20细胞在细胞因子的表达方面有无差异、在体内成瘤组织中A20与LV-mCD99L2-A20分别对细胞因子的表达产生何种影响、LV-mCD99L2-A20体内外的细胞因子表达能否有助于HL动物模型的构建等一系列问题有待解决,因此在分别构建了A20/LV-mCD99L2-A20动物模型后本研究将从细胞因子表达的角度初步探讨A20/LV-mCD99L2-A20细胞模型和动物模型与HL之间的关系。
目的
1、鉴定低表达mCD99L2的LV-mCD99L2-A20细胞亚系,以探究mCD99L2基因在HL的H/RS细胞形成中的作用,并为后续模型动物的构建奠定基础。
2、鼠源性B淋巴瘤细胞株A20以不同方式接种同源BALB/c小鼠,构建有免疫功能的鼠B淋巴瘤动物模型,探讨不同造模方式的优劣,为本研究后续工作奠定基础,并为B淋巴瘤相关研究提供适合的实验平台。
3、将LV-mCD99L2-A20细胞亚系接种BALB/c小鼠,进行成瘤情况观察、病理形态学观察、分子生物学鉴定和免疫标记的检测,探讨成瘤特点、成瘤组织病变特征与A20动物模型之间的差异及与HL之间的关系。
4、借助生物信息学软件对HL与细胞因子相关性进行检索和分析。利用蛋白芯片初步检测A20/LV-mCD99L2-A20细胞和BALB/c成瘤组织中细胞因子的表达、及差异,寻找LV-mCD99L2-A20细胞模型、动物模型与构建类人HL动物模型之间关系的相关依据。
方法
1、LV-mCD99L2-A20细胞亚系的鉴定
对前期构建的慢病毒质粒稳定干扰的LV-mCD99L2-A20克隆株进行体外培养,倒置显微镜、HE染色观察LV-mCD99L2-A20细胞的形态变化并进行计数,随机选择多代细胞进行DNA-PCR检测ShRNA干扰载体的整合,RT-PCR及荧光定量PCR检测目的基因mCD99L2的表达水平,免疫荧光观察mCD30分子标记,MTT法观察细胞体外增殖能力,流式细胞仪检测细胞周期及细胞的免疫表型(CD3、CD4、CD8、CD19、CD20、CD30)。
2、五种方式构建有免疫功能A20鼠B淋巴瘤动物模型
将鼠源性B细胞淋巴瘤细胞株A20经皮下接种、尾静脉注射、脾脏注射、腹腔注射于同源BALB/c小鼠和A20接种裸鼠成瘤组织移植BALB/c小鼠,观察动物成瘤时间、成瘤率、皮下肿瘤生长速度、内脏成瘤播散部位;取动物脏器行石蜡包埋、制备病理切片、HE染色观察病理学形态;运用流式细胞仪检测其瘤组织细胞悬液中CD3、CD4、CD8、CD19、CD30的表达;流式细胞仪检测正常BALB/c小鼠、接种成瘤小鼠和未成瘤小鼠外周血中淋巴细胞亚群的比例。
3、LV-mCD99L2-A20鼠淋巴瘤动物模型的构建
将经过鉴定的低表达mCD99L2基因的LV-mCD99L2-A20细胞亚系经皮下接种、尾静脉注射和裸鼠成瘤组织块移植的方法接种BALB/c小鼠,观察动物成瘤情况;取动物脏器行石蜡包埋、病理切片、HE染色观察病理学形态;计数成瘤切片中H/RS样细胞;免疫荧光检测瘤组织mCD30分子标记;运用流式细胞仪检测其瘤组织细胞悬液中CD3、CD4、CD8、CD19、CD30的表达。肿瘤组织进行原代培养,DNA-PCR检测载体的整合,RT-PCR检测mCD99L2基因mRNA水平。流式细胞仪检测小鼠外周血和脾脏中淋巴细胞亚群的比例。
4、A20/LV-mCD99L2-A20体内外细胞因子表达蛋白芯片初步检测
运用生物信息学软件GeneClip对HL和62种细胞因子进行文献检索,构建网络关系图。运用小鼠细胞因子功能分类蛋白芯片(RayBiotech mouse cytokineAntibody Arrays)对A20细胞和LV-mCD99L2-A20细胞、A20细胞BALB/c皮下成瘤组织和LV-mCD99L2-A20细胞BALB/c鼠皮下成瘤组织进行蛋白质抽提、蛋白质定量,膜封闭和孵育、62种小鼠细胞因子检测、图像采集和数据分析,对差异细胞因子进行初步分析。
结果
1、LV-mCD99L2-A20细胞亚系的鉴定
(1)传代过程中倒置显微镜观察和HE染色均发现LV-mCD99L2-A20细胞中存在类似H/RS细胞的大细胞。随机抽取A、B、C、D代LV-mCD99L2-A20细胞计数其中的大细胞发现较A20细胞中的大细胞多,差异有显著性(P＜0.05)。
(2)抽提多代LV-mCD99L2-A20细胞DNA进行PCR均能检测出ShRNA干扰载体稳定整合至A20细胞基因组。
(3) RT-PCR及荧光定量PCR检测目的基因mCD99L2均较A20细胞呈低水平表达,干扰效率约为50%。
(4) MTT检测发现LV-mCD99L2-A20细胞体外增殖能力较对照A20组和空载体组减慢,差异有显著性(P＜0.05)。
(5)流式细胞仪检测细胞周期发现LV-mCD99L2-A20细胞S期比例与A20细胞无显著性差异(P＞0.05),但是G2期较A20细胞长,差异有显著性(P＜0.05)。
(6)免疫荧光检测LV-mCD99L2-A20细胞及其中的大细胞CD30呈阳性表达。
(7) LV-mCD99L2-A20细胞的CD3表达(7.47±1.27%)较A20细胞(18.93±3.87%)降低,CD8表达(8.33±3.89%)较A20细胞(18.27±3.45%)降低,CD20表达(13.10±5.16%)较A20细胞(35.33±2.25%)降低,CD30表达(76.0±12.44%)较对照A20细胞(41.70±2.60%)升高,差异有显著性(n=3,P＜0.05),而CD4、CD19差异不显著(P＞0.05)。空载体组与对照A20细胞CD抗原表达无显著差异(P＞0.05)。
2、五种方式构建有免疫功能A20鼠B淋巴瘤动物模型
(1)皮下成瘤:BALB/c小鼠皮下注射2×10~5组(n=7)未成瘤,2×10~6组(n=7)、2×10~7组(n=7)和裸鼠瘤组织移植BALB/c小鼠组(n=7)成瘤率皆为100%,成瘤时间分别为15.29±3.2天、7.0±0.82天、6.29±0.49天。
(2)内脏成瘤:BALB/c小鼠尾静脉注射2×10~6组、2×10~7组、脾脏注射组、腹腔注射组成瘤率分别为71.4%(5/7)、100%(7/7)、71.4%(5/7)、14.3%(1/7),成瘤时间分别为76.8±12.0天、26.1±7.99天、32.6±5.99天和27天。尾静脉成瘤部位播及肝脏、脾脏、胰腺、肾脏、食道、胃、肠、肠系膜、脑、淋巴结、骨、子宫、肌肉等多脏器和组织。
(3) BALB/c鼠A20成瘤组织镜下观察:瘤细胞呈弥漫性分布,大小一致,细胞核大、圆形或不规则形,核仁明显,胞质中等量、嗜碱性,类似人弥漫大B细胞淋巴瘤。
(4)流式细胞仪检测成瘤组织细胞悬液(n=8)CD3、CD4、CD8、CD19、CD30的表达阳性细胞比例分别为(%):49.27±23.75、6.07±3.65、51.2±23.1、67.06±16.39、37.93±17.03。
(5)流式细胞仪检测外周血淋巴细胞亚群:各组间CD3、CD4阳性细胞比例存在显著性差异。接种A20细胞成瘤BALB/c小鼠与正常BALB/c小鼠相比CD3(P=0.014)、CD4(P=0.009)阳性细胞比例下降,差异有显著性。接种A20细胞成瘤BALB/c小鼠与接种A20细胞未成瘤BALB/c小鼠相比CD3(P=0.004)、CD4(P=0.006)阳性细胞比例下降,差异显著。接种A20细胞未成瘤BALB/c小鼠与正常BALB/c鼠相比CD3(P=0.592)、CD4(P=0.867)、CD8(P=0.398)、CD19(P=0.510)阳性细胞比例和CD4/CD8(P=0.545)比值差异皆不显著。
3、LV-mCD99L2-A20鼠淋巴瘤动物模型的构建
(1) LV-mCD99L2-A20接种后成瘤情况:皮下接种2×10~7细胞于4只裸鼠(6个位点),100%成瘤,平均成瘤时间为13.33±4.63天,生长速度较A20裸鼠皮下接种2×10~7组生长缓慢。皮下注射2×10~7LV-mCD99L2-A20细胞于BALB/c小鼠14只,7.3%(1/14)成瘤;成瘤时间10天。裸鼠瘤组织移植BALB/c小鼠14只,7.3%(1/14)成瘤,成瘤时间6天。BALB/c小鼠尾静脉注射组28只,三月宰杀时均未成瘤。
(2)病理形态:裸鼠和BALB/c小鼠成瘤组织光镜观察瘤细胞大小不一,弥漫分布,可见散在的胞浆丰富的大细胞,呈双核、多核或不规则核,核仁大,极其类似人HL的H/RS细胞形态。BALB/c小鼠H/RS细胞周围存在一定量淋巴细胞的浸润;瘤组织流式检测CD3、CD4、CD8、CD19和CD30皆呈较高表达。
(3) BALB/c小鼠瘤组织原代培养:DNA-PCR检测LV-mCD99L2干扰载体整合在细胞基因组,RT-PCR显示mCD99L2基因mRNA水平较A20细胞为低。
(4)流式细胞仪检测外周血淋巴细胞亚群:各组间CD3、CD4、CD8、CD19阳性细胞比例存在显著差异。接种A20细胞未成瘤BALB/c小鼠与正常BALB/c小鼠各指标差异无显著性(P＞0.05)。接种LV-mCD99L2-A20细胞未成瘤BALB/c小鼠与正常BALB/c小鼠相比外周血CD3(P=0.014)、CD4(P=0.018)阳性细胞比例下降而CD19(P=0.012)阳性细胞比例升高,差异有显著性;而CD8(P=0.075)阳性细胞比例差异不显著。接种LV-mCD99L2-A20细胞未成瘤BALB/c小鼠与接种A20细胞未成瘤BALB/c小鼠相比CD3(P=0.004)、CD4(P=0.012)和CD8(P=0.007)阳性细胞比例下降,差异有显著性;而CD19(P=0.052)阳性细胞比例差异不显著。
(5)脾脏淋巴细胞亚群:接种A20细胞未成瘤BALB/c小鼠与正常BALB/c惺笙啾菴D3(P=0.003)、CD4(P=0.007)、CD8(P=0.000)阳性细胞比例下降,CD19(P=0.004)阳性细胞比例和CD4/CD8(P=0.000)比值升高,差异有显著性。接种LV-mCD99L2-A20细胞未成瘤BALB/c小鼠与正常BALB/c小鼠相比CD3(P=0.000)、CD4(P=0.001)、CD8(P=0.000)阳性细胞比例下降而CD19(P=0.000)阳性细胞比例和CD4/CD8(P=0.000)比值升高,差异有显著性。接种LV-mCD99L2-A20细胞未成瘤BALB/c小鼠与接种A20细胞未成瘤BALB/c小鼠相比CD3(P=0.009)、CD4(P=0.020)阳性细胞比例下降,CD19(P=0.020)阳性细胞比例升高,差异有显著性;CD8(P=0.170)阳性细胞比例和CD4/CD8(P=0.646)比值无显著性差异。
4、A20/LV-mCD99L2-A20细胞和组织细胞因子表达的蛋白芯片初步检测
(1) LV-mCD99L2-A20细胞较A20细胞上调(≥1.5倍)的细胞因子有CD30T、IL-12p40/p70、IL-3、IFNγ、CXCL16、MIP-1α、CD40,其中CD30T表达上调2.91倍。未见表达下调超过1.5倍的细胞因子表达。
(2)LV-mCD99L2-A20细胞BALB/c小鼠成瘤组织较LV-mCD99L2-A20细胞上调≥1.5倍的细胞因子主要有VCAM-1、MIP-1γ、MIG、IL-10、RANTES、PF-4、CXCL16、VEGF、MIP-3α、P-Selectin,其中VCAM-1、MIP-1γ、MIG、IL-10上调≥2倍;下调≥1.5倍的细胞因子主要有IL-6、Eotaxin、TNFα、TIMP-1、SCF、IFNγ、IL-3、CD30T、IL-5、IGFBP-6、TECK、L-Selectin、Fas Ligand、IL-3 Rb、IL-9,其中IL-6下调3倍以上。
(3)LV-mCD99L2-A20细胞接种BALB/c小鼠成瘤组织较A20细胞接种BALB/c小鼠成瘤组织中下调(≥1.5倍)的细胞因子如Eotaxin、IGFBP-6、IL-12 p40/p70、IGFBP-5、MIP-1α、KC、CXCL16,其中Eotaxin、IGFBP-6下调超过2倍;而上调(≥1.5倍)的细胞因子仅是VEGF。
结论
1、LV-mCD99L2-A20细胞亚系中存在类似人H/RS细胞形态的H/RS样细胞,LV-mCD99L2-A20细胞在增殖能力、细胞周期和免疫表型方面较类似人HL。
2、A20细胞BALB/c小鼠皮下移植瘤模型成瘤时间短、尾静脉接种式成的血行播散性模型成瘤部位广泛,均为研究有免疫功能鼠B淋巴瘤适宜的实验动物模型。
3、LV-mCD99L2-A20细胞较A20细胞在BALB/c鼠难以成瘤,但在成瘤组织中出现似人HL的H/RS样细胞,伴有一定程度的背景淋巴细胞的浸润。
4、LV-mCD99L2-A20细胞较A20细胞上调了一些与报道中的HL或H/RS细胞相关的细胞因子;LV-mCD99L2-A20细胞与A20细胞在BALB/c小鼠体内成瘤组织中细胞因子表达不同。
创新之处
本研究从体外实验、裸鼠成瘤实验和BALB/c鼠成瘤实验三个水平,从形态学、免疫表型、生物学行为三个主要方面并结合小鼠免疫功能初步检测和细胞因子表达蛋白芯片初步检测,较为系统地对比了A20细胞与LV-mCD99L2-A20细胞在细胞模型之间、动物模型之间的差异,揭示了mCD99L2基因低表达对体外、体内诱导H/RS样细胞具有重要作用;提示了LV-mCD99L2-A20 BALB/c鼠模型在出现H/RS样细胞的同时其良好的预后可能与HL具有某种相似性;结合体内外细胞因子表达的差异分析了LV-mCD99L2-A20细胞模型、动物模型与构建HL之间的相关性,为探索性的研究,为构建稳定的HL动物模型奠定了坚实的基础。
Backgroud
Lymphomas are immune system malignant neoplasms of cells native to lymphoid tissue.Along with the wide use of immunosuppressant for organ transplant, AIDS occurence,and chemotherapy for tumors,the occurrences of lymphomas have drastically increased in recent years.
Different from non-Hodgkin's lymphoma(NHL),Hodgkin's lymphoma(HL) is a peculiar form of malignancy in which the clonal B cell population,the so called Hodgkin/Reed-Sternberg(H/RS) cells and their variants,is responsible for less than 1%of the bulk of the tumor.The mechanism of development of HL and H/RS cell is still illusive.The relationship between H/RS cell and background cells is also restricted for lack of suitable animal model which mimics the characteristics of HL. There are reports that human H/RS cell is formed for the low expression of CD99 (MIC2).Our research work have previously transfected the mouse B lymphoma cell line A20,which expressed mouse CD99 antigen-like 2 gene,using Letivirus ShRNA vector and constructed subseries of A20 cell line with low mCD99L2 gene expression,named "LV-mCD99L2-A20".Among the LV-mCD99L2-A20 cells,giant cells like human HL H/RS cells were found.Whether these giant cells could consistently present during continuous passage culture of LV-mCD99L2-A20 cells and what are the immunophenotypes of these cells? How about the tumors induced by LV-mCD99L2-A20 cells in the nude mouse and BALB/c mouse? What's the difference between LV-mCD99L2-A20 cells and A20 cells in mouse with nomal immune system?These problems need to be investigated.
The animal model people often used were engrafted immunodeficiency animals inoculated with lymphoma cell lines or the tissue from lymphoma patients,which were of great importance during the research into molecular mechanisms of lmphomas.Most of them are immunodeficiency animals.Lymphoma is a kind of tumor envolved with body immune system.For lack of T lymphocyte or/and B lymphocyte,neither the nude mouse nor the severe combined immunodeficiency mouse could mimic the immune enviroment of human body and it is hard to use these models to reveal the interaction between lymphoma and immune system of human body.Therefore these animal models are limited in the research of clinic therpy and drug usage.
The abundant reactive cells in HL were related not only with whole immune system of the body,but the immuno-microenviroment induced by H/RS cells.It has been postulated that the presence of abundant reactive cells in HL tissue is due to the production of several kinds of cytokines and/or chemokines.Really what is the relationship between HL H/RS cell and cytokines? Is there any difference in the cytokines' expression between A20 cells and LV-mCD99L2-A20 cells? What kinds of cytokines will be induced in BALB/c mouse by A20 cells and LV-mCD99L2-A20 cells respectively? Will the secretion of cytokines in LV-mCD99L2-A20 cells and tumor tissues benefit the construction of real HL animal model? After the costruction of A20/LV-mCD99L2-A20 animal model,our work will discuss the relationship between LV-mCD99L2-A20 cell model and/or animal model and costruction of HL.
Objective
1.Identifying the subseries which express low level of mouse CD99 antigen-like 2 gene(mCD99L2) of mouse B lymphoma cell line(A20),named LV-mCD99L2-A20, so as to investigate the importance of mCD99L2 in the formation of Hodgkin/ Reed-Sternberg(H/RS) like cell.
2.Inoculating mouse B lymohoma cell line A20 to homologic BALB/c mouse in different ways,so as to construct mouse B lymphoma animal models and compare the difference between them,thus providing suitable animal models for research in B cell lymphoma.
3.Using LV-mCD99L2-A20 cells to inoculate BALB/c mouse,investigating the tumor formation,observing its pathological characteristics,identifying its molecular biology and detecting its immunophenotypes so as to compare the biological difference between in vivo and in vitro,comparing the pathological characteristics between LV-mCD99L2-A20 cell-induced tumors and A20 cell-induced tumors,thus analyzing the relationship between the difference and human HL.
4.Applying the software of bioinformatics to retrieve and analyze the relationship between HL and cytokines.Using mouse cytokine antibody arrays to investigate the expression of cytokine in the cells and tumor tissues,comparing its difference and analyzing the relationship between the diffenrence and construction of human HL like animal model,thus providing experimental data for our profound research work.
Method
1.During the period of continuous passage culture of A20 cells formerly transfected with LentiVirus-mCD99L2 vector,named LV-mCD99L2-A20 cells,choosing cells of different passages respectively:examining the integrated status of vector using DNA-PCR;analyzing RNA interference efficiency by RT-PCR and real time RT-PCR, observing morphological characteristics and counting numbers of giant cells under light microscope,detecting expression of mouse CD30 molecules in LV-mCD99L2 -A20 cells by immunofluorenscence,observing the growth curve by MTT and detecting the cell cycle and immunophonotypes of A20 cells and LV-mCD99L2-A20 cells such as CD3,CD4,CDS,CD19,CD20,CD30.
2.Innoculating mouse B lymohoma cell line A20 to homological BALB/c mouse by subcutaneous inoculation,intravenous injection,spleen inoculation,intraperitoneal injection and transplantation of tissue from nude mouse,observing the tumor formation,paraffin imbedding the organs of animals,pathological section and HE staining to observe its pathological characteristics.Detecting CD3,CD4,CD8,CD19, CD30 expression of the cell suspension of tumor tissues with Fluorescence Activated Cell Sorter(FACS) technology.Comparing the difference of lymphocyte subpopulation in normal mouse and mouse with or without tumors.
3.Using identified LV-mCD99L2-A20 cells to inoculate homologic BALB/c mouse by subcutaneous inoculation and intravenous injection,observing the tumor formation,paraffin imbedding the organs of animals,pathological section and HE staining to observe its pathological characteristics,detect the expression of CD3,CD4, CD8,CD19,CD30 in cell suspension of tumor tissues by FACS and also compare the difference of lymphocyte subpopulation in peripheral blood and spleen of normal mouse and mouse without tumors after A20 inoculation and LV-mCD99L2-A20 inoculation.
4.Using bioinformatics software Geneclip to retrieve the articles about the HL and/or H/RS cell and 62 kinds of cytokines,analyzing the relationship between them.Using RayBiotech mouse cytokine Antibody Arrays to investigate the expression of cytokines in A20 and LV-mCD99L2-A20 cells and their related tumor tissues, extracting and quantitating proteins,sealing and incubating membranes,detecting expressions of cytokines,imaging photos and analyzing the difference between them.
Result
1.Identification of subseries of A20 with low expression of mCD99L2 gene (LV-mCD99L2-A20):
(1) Giant cells like Hodgkin/Reed-Sternberg cells(H/RS-like cell) were found during continuous passage culture of LV-mCD99L2-A20 cells.The number of giant cells in A,B,C,D passage cells were more than those in A20 cell line,with dramatic difference(P＜0.05)
(2) During continuous passages of LV-mCD99L2-A20 cells,282 bp fragments of LV-mCD99L2 ShRNA vector were detected by DNA extraction and following PCR reaction.
(3) Expression of mCD99L2 gene mRNA in LV-mCD99L2-A20 cells was lower (about 50%)than those in A20 control group revealed by RT and real-time PCR.
(4) The proliferating ability of LV-mCD99L2-A20 cell deteced by wethod of MTT is slower than the contral A20 group and mock group,with dramatic difference (P＜0.05).
(5) Cell cycle detection by FACS revealed that S phase of each group is of no significant difference(P＞0.05),while the G2 phase is longer than the contral A20 group and mock group,with significent difference(P＜0.05).
(6) LV-mCD99L2-A20 cells,including giant cells,were positive labeled by mouse CD30 antibody in immunofluorenscence test.
(7) The CD3 expression of LV-mCD99L2-A20 cell(7.47±1.27%)is lower than A20 cell(18.93±3.87%),CD8 expression of LV-mCD99L2-A20 cell(8.33±3.89 %)is lower than A20 cell(18.27±3.45%),CD20 expression of LV-mCD99L2-A20 cell(13.10±5.16%)is also lower than A20 cell(35.33±2.25%),while CD30 expression of LV-mCD99L2-A20 cell(76.0±12.44%)is higher than A20 cell (41.70±2.60%),all with significent difference(n=3,P＜0.05).The CD4 and CD19 expression of LV-mCD99L2-A20 cell were of no significant difference with A20 cell (P＞0.05).The mock group is of no significant difference with A20 cell in each CD antigen ex[ression(P＞0.05).
2.Construction of animal model using mouse B lymphoma A2O cell line using different methods:
(1) There are no tumor formed in 2×10~5 BALB/c mouse group,while 100%tumor formation in the BALB/c mouse of subcutaneous inoculation(2×10~6 and 2×10~7) groups and tissue(from nude mouse) trans-plantation group after inoculating about 15.29±3.2 days,7.0±0.82 days and 6.29±0.49 days,respectively. (2) There are 71.4%(5/7) and 100%(7/7) tumor formation in intravenous injection (2×10~6 and 2×10~7) group respectively,71.4%(5/7) in spleen inoculation group and 14.3%(1/7) in intraperitoneal injection group after about 76.8±12.0 days,26.1±7.99 days,32.6±5.99 days and 27 day.The tumor formed in several organs of mouse,such as liver,spleen pancrease,kidney,esophagus,stomach,intestine,mesentery,brain, lymphocytic node,bone,uterus and muscles et al.
(3) The tumor cells diffused with large round or irregular nucleus in the tissues, nucleoli is obvious and cytoplasm is light staining,somewhat like diffuse large B cell lymphoma(DLBCL).
(4) The expression of CD3、CD4、CD8、CD19、CD30 in the cells from tumor tissues(%) are 49.27±23.75、6.07±3.65、51.2±23.1、67.06±16.39 and 37.93±17.03, respetively.
(5) FACS detection of proportions of lymphocyte subsets in peripheral blood show significant differences in CD3 and CD4 expressions between each groups.The proportions of lymphocyte subsets in the blood of mouse with tumor genesis after A20 cell inoculation are lower in CD3(P=0.014) and CD4(P=0.009) expression than those of normal BALB/c mouse and also lower in CD3(P=0.004) and CD4(P=0.006) expression than those in mouse without tumor genesis after A20 cell inoculation.The proportions of lymphocyte subsets in the blood of mouse without tumor genesis after A20 cell inoculation are of no significant difference from those of normal BALB/c mouse:CD3(P=0.592),CD4(P=0.867),CD8(P=0.398),CD19(P=0.510) and CD4/CD8 (P=0.545) ratio.
3.Construction of animal model using identified LV-mCD99L2-A20 sbseries:
(1) There are 100%tumor formed in the nude mouse of subcutaneous inoculation (2×10~7) group after average about 13.33±4.63 days,while only 7.1%(1/14) tumor formed in the BALB/c subcutaneous inoculation(2×10~7) group and nude tumor tissues transplanting group,after 10 days and 6 days,respectively.Intravenously inoculated BALB/c mouse group(n=28) has no tumor formed till three months after inoculation.
(2) Tumor cells in nude mouse and BALB/c mouse indicated different sizes under light microscope,diffuse contribution,large and deep stained nucleus,sparse binucleated,polynucleated observation,which was similar to pathological characteristics of human HL H/RS cells.Besides,lymphocytes infiltrated into tumor tissues of BALB/c mouses,with high expression of CD3+ T lymphocyte,CD4+ T lymphocyte,CD8+ T lymphocyte and high CD30 expression.
(3) During primary culture of tumor tissues,282 bp fragments of ShRNA vector were detected in LV-mCD99L2-A20 B1 cells.Expression of mCD99L2 gene mRNA in LV-mCD99L2-A20 B1 cells was lower than A20 control group revealed by RT-PCR.
(4) FACS detection of proportions of lymphocyte subsets in peripheral blood show significant differences in CD3,CD4,CD8 and CD19 expressions between each groups.The proportions of lymphocyte subsets in the blood of mouse without tumor genesis after LV- mCD99L2-A20 cells inoculation are of significant differences from those of normal BALB/c mouse--lower in CD3(P=0.014),CD4(P=0.018) and higher in CD19(P=0.012) expression,while are of no significant differences in CD8(P=0.075).The proportions of lymphocyte subsets in the blood of mouse without tumor genesis after LV- mCD99L2-A20 cells inoculation are of significant differences from those of A20 inoculated BALB/c mouse -- lower in CD3(P=0.004), CD4(P=0.012) and CD8(P=0.007) expression while no significant differences in CD19(P=0.052) expression.
(5) FACS detection of proportions of lymphocyte subsets in spleen show significant differences between each groups.The proportions of lymphocyte subsets in the spleen of mouse without tumor genesis after LV-mCD99L2-A20 cells inoculation are of significant differences from those of normal BALB/c mouse --lower in CD3 (P=0.000),CD4(P=0.001),CD8(P=0.000) expressions while higher in CD19(P=0.000) expressions and CD4/CD8(P=0.000) ratio.The proportions of lymphocyte subsets in the spleen of mouse without tumor genesis after LV-mCD99L2-A20 cells inoculation are of significant differences from those of A20 inoculated BALB/c mouse - lower in CD3(P=0.009),CD4(P=0.020) expressions,higher in CD19(P=0.020) expression, while no significant differences in CD8(P=0.170) and CD4/CD8(P=0.646) ratio.
4.Expression of cytokines in A20 / LV-mCD99L2-A20 cells and tissues:
(1) The up-regulated cytokines(≥1.5 folds) in LV-mCD99L2-A20 cells,when compared with A20 cells,include CD30T,IL-12p40/p70,IL-3,IFNγ,,CXCL16, MIP-1αand CD40,among which CD30T is up-regulated by 2.91 folds.There is no down-regulated(≥1.5 folds) cytokines in LV-mCD99L2-A20 cells compared with A20 cells.
(2) The up-regulated cytokines(≥1.5 folds) in LV-mCD99L2-A20 cell-induced tumor in BALB/c mouse,compared with those in LV-mCD99L2-A20 cells,include VCAM-1,MIP-1γ,MIG,IL-10,RANTES,PF-4,CXCL16,VEGF,MIP-3αand P-Selectin,among which VCAM-1,MIP-1γ,MIG and IL-10 up-regulated in excess of two folds.The down-regulated cytokines(≥1.5 folds) include IL-6、Eotaxin、TNFα、TIMP-1,SCF,IFNγ,IL-3,CD30 T,IL-5,IGFBP-6,TECK,L-Selectin,Fas Ligand,IL-3 Rb and IL-9,among which IL-6 down-regulated in excess of three folds.
(3) When compared with A20 cell-induced tumor,the down-regulated cytokines(≥1.5 folds) include Eotaxin,IGFBP-6,IL-12 p40/p70,IGFBP-5,MIP-1α,KC and CXCL16 in LV-mCD99L2-A20 cell-induced tumor in BALB/c mouse,among which Eotaxin and IGFBP-6 down-regulated in excess of two folds,while the up-regulated cytokines(≥1.5 folds) is only VEGF.
Conclusion
1.There exist giant cells with morphologic characteristics that like human HL H/RS cells in the subseries of A20 which show low expression of mCD99L2 gene(LV-mCD99L2 -A20 cell).The biological characteristics and immunophotypes of LV-mCD99L2-A20 cell much more mimics HL than A20 cell.
2.The subcutaneously transplanted tumors of BALB/c mouse are formed in a shorter time period after A20 cell inoculation than those in other animal models.The hematogenous disseminated animal model by more than 10~6 A20 cell inoculation is involved with several visceral organs.These animal models provide suitable animal models for research of B lymphomas in mouse with nomal immune function.
3.LV-mCD99L2-A20,when inoculated into BALB/c mouse,has lower tumor genesis compared with A20 inoculation,while more giant cells with the morphologic characteristics that like human HL H/RS cells dispersed in LV-mCD99L2-A20 cell-induced tumors than in A20-induced tumors in BALB/c mouse,accompanied by some infiltrating lymphocytes.
4.LVomCD99L2-A20 cell is more likely to express cytokines usually appeared in HL cell lines or in H/RS cells.There exist differences between the cytokine expressions of LV-mCD99L2-A20 cell-induced tumors and A20-induced tumors in BALB/c mouse.
New point
From three levels:in vitro investigation,nude mouse and BALB/c mouse in vivo investigation,the research compared the fifferences systematically between A20 and LV-mCD99L2-A20 cell models,between their animal models by morphological observation,detection of immunophonotypes and biological characteristics combining with primary investigation of immune function and cytokines expressions.The results revealed and confirmed that the low expression of mCD99L2 gene is of great importance to the construction of H/RS-like cell model in vitro and in vivo and also revealed a better prognosis of LV-mCD99L2-A20 cells than A20 cells in animal with normal immune function,which may be some similar to genesis of HL. Through primary detection of cytokines in vitro and in vivo,analyzing the relationship between LV-mCD99L2-A20 cell model,animal model and construction of HL animal model.All of these made a better basis for prolonged construction of HL animal model.
引文
1. Kuppers R and Hansmann ML. The Hodgkin and Reed/Sternberg cell. IJBCB, 2005,37(3):511-517.
2. Kuppers R. B cells under influence: transformation of B cells by Epstein-Barr virus. Nat Rev Immunol, 2003,3(10):801-12.
3. Zongli Qi, Tong Zhao, Xinhua Zhou, et al. EBER-1, ISH, LMP1 Immuno- histochemical tests and PCR for detecting Epstein-Barr Virus from Hodgkin's lymphoma tissues in southern China. U.S.Chinese Journal of Lymphology and Oncology, 2004,3 (1):5-10.
4. Kang LC, Dunphy CH.Immunoreactivity of M1C2 (CD99) and terminal deoxynucleotidyl transferase in bone marrow clot and core specimens of acute myeloid leukemias and myelodysplastic syndromes. Arch Pathol Lab Med. 2006, 130(2): 153-7.
5. Dworzak MN, Fritsch G, Fleischer C, et al .CD99 (MIC2) expression in paediatric B-lineage leukaemia/lymphoma reflects maturation- associated patterns of normal B-lymphopoiesis. Br J Haematol. 1999, 105(3):690-5.
6. Hystad ME, Myklebust JH, Bo TH, et al .Characterization of early stages of human B cell development by gene expression profiling. J Immunol. 2007, 179 (6):3662-71.
7. Miyagawa Y, Okita H, Nakaijima H, et al.Inducible expression of chimeric EWS/ETS proteins confers Ewing's family tumor-like phenotypes to human mesenchymal progenitor cells. Mol Cell Biol. 2008 Jan 22 [Epub ahead of print]
8. Dworzak, MN, Froschl, G, Printz, D, et al. CD99 expression in T-lineage ALL: implications for flow cytometric detection of minimal residual disease [J]. Leukemia, 2004, 18(4):703-8.
9. Shapiro M, Wasik MA, Junkins-Hopkins JM, et al. Complete remission in advanced blastic NK-cell lymphoma/leukemia in elderly patients using the hyper-CVAD regimen. Am J Hematol.2003, 74(1):46-51.
10. Odashiro AN, Leite LV, Oliveira RS, et al. Primary orbital mesenchymal chondrosarcoma:a case report and literature review. Int Ophthalmol. 2008 Jan 10 [Epub ahead of print]
11. Diwan AH, Skelton HG 3rd, Horenstein MG, et al.Dermatofibrosarcoma protuberans and giant cell fibroblastoma exhibit CD99 positivity. J Cutan Pathol. 2008 Jan 14 [Epub ahead of print]
12.Ramsay AD,Bates AW,Williams S,et al.Variable antigen expression in hepatoblastomas.Appl Immunohistochem Mol Morphol.2008 Jan 25[Epub ahead of print]
13.Park CK,Shin YK,Kim TJ,et aI.High CD99 expression in memory T and B cells in reactive lymph nodes.J Korean Med Sci.1999,14(6):600-6.
14.Manara MC,Bernard G,Lollini PL,et al.CD99 acts as an oncosuppressor in osteosarcoma [J].Mol Biol Cell,2006,17(4):1910-21.
15.Imbert AM,Belaaloui G,Bardin F,et ai.CD99 expressed on human mobilized peripheral blood CD34+ cells is involved in trans- endothelial migration[J].Blood,2006,108(8):2578-86.
16.Zucchini C,Rocchi A,Manara MC,et al.Apoptotic genes as potential markers of metastatic phenotype in human osteosarcoma cell lines.Int J Oncol.2008,32(1):17-31.
17.Lee JH,Kim SH,Wang LH,et al.Clinical significance of CD99 down- regulation in gastric adenocarcinoma.Clin Cancer Res.2007,13(9):2584-91.
18.Scotlandi K,Zuntini M,Manara MC,et al.CD99 isoforms dictate opposite functions in tumour malignancy and metastases by activating or repressing c-Src kinase activity.Oncogene.2007,26(46):6604-18.
19.Byun H J,Hong IK,Kim E,et ai.A splice variant of CD99 increases motility and MMP-9expression of human breast cancer cells through the AKT-,ERK-,and JNK-dependent AP-1 activation signaling pathways.J Biol Chem.2006,281(46):34833-47.
20.Lee IS,Kim SH,Song HG,et al.The molecular basis for the generation of Hodgkin and Reed-Sternberg cells in Hodgkin's iymphoma.Int J Hematol.,2003,77(4):330-5.
21.Sub YH,Kim MK,Shin YK,et al.Mutations of the immunoglobulin heavy chain variable region gene in CD99-deficient B JAB cells line.Mol Cells,2002,13:237 -244.
22.李先茂,李燕,赵彤,等.霍奇金淋巴瘤CD99基因表达缺失的意义.第四军医大学学报,2004.25(23):2136-2137.
23.沈丽佳,何滢,蒋会勇,等.mic2/CD99在经典型霍奇金淋巴瘤H/RS细胞中的表达及与Eber-1/LMP-1相关性的研究.中国病理生理杂志,2006,22(4):776-780.
24.何滢,硕士论文.L428细胞CD99基因上调H/RS细胞形态转化及免疫表型的研究.
25.Suh YH,Shin YK,Kook MCH,et al.Cloning,genomic organization,alternative transcripts and expression analysis of CD99L2,a novel paralog of human CD99,and identification of evolutionary conserved motifs.Gene,2003,307:63-76.
26.Gabriele B,Stephan K,Stefan B,et al.Mouse CD99 participates in T-cell recruitment into inflamed skin.Blood.,2004,104:3205-3213.
27.Schenkel AR,Dufour EM,Chew TW,et al.The murine CD99-related molecule CD99-1ike 2(CD99L2) is an adhesion molecule involved in the inflammatory response.Cell Commun Adhes.2007,14(5):227-37.
28.van Wanrooij EJ,de Vos P,Bixel MG,et al..Vaccination against CD99 inhibits athero genesis in low-density lipoprotein receptor-deficient mice.Cardiovasc Res.2008 Mar 3[Epub ahead of print]
29.沈丽佳,方唯意,谢思明,等.小鼠B淋巴瘤A20细胞株mCD99L2基冈表达检测及真核表达载体的构建.南方医科大学学报,2006,26(2):144-149.
30.何滢,沈丽佳,李祖国,等.mCD99L2基因沉默对小鼠B淋巴瘤细胞系A20细胞转化为H/RS样细胞的影响.基础医学与临床,2007,27(6):616-620.
31.Hayashi K,Teramoto N,Akagi T.Animal in vivo models of EBV-associated lympho -proliferative disease:special references to rabbit models[J].Histo Histopathol,2002,17(4):1293-1310.
32.Burgess S C,Young J R,Baaten B J,et al.Marek's disease is a natural model for lymphomas over-expressing Hodgkin's disease antigen[J].PNAS,2004,101(38)..13879-13884.
33.Osterrieder N,Kamil J P,Schumacher D,Tischer B K,Trapp S.Marek's disease virus:from miasma to model.Nat Rev Microbiol,2006,4(4).- 283-294.
34.Hasegawa H,Sawa H,Lewis M J,et al.Thymus-derived leukemia-lymphoma in mice transgenic for the Tax gene of human T-lymphotropic virus type Ⅰ.Nat Med,2006,12(4):466-472.
35.Nilsson J A,Nilsson L M,Keller U,et al.Id_2 is dispensable for myc-induced lymphoma genesis[J].Cancer Res,2004,64(20):7296-7301.
36.Beverly W B,John A,Anthony M,et al.The human BCL-6 transgene promotes the development of lymphomas in the mouse[J].PNAS,2004,101(39):14198-14203.
37.Brandt J VD,Kwon S H,McPherson K G,et al.Unexpected features of acute T lymphoblastic lymphomas in NotchlIC transgenic rats.Eur J Immunol,2006,36(8):2223-2234.
38.Martin W,Ulrike S,Andreas B,et al.3-[~(18)F]Fluoro-3'-Deoxythymidine([~(18)F]) as positron emission tomography tracer for imaging proliferation in a murine B-cell lymphoma model and in the human disease[J].Cancer Res,2003,63:2681-2687.
39.邵晓枫,杨纯正,熊冬生,等.人B淋巴瘤裸小鼠腹腔内移植模型[J].中国肿瘤临床,2001,28(3):217-219.
40.刘秋珍,脱朝伟,张宁,等.人小肠原发性恶性淋巴瘤裸鼠原位移植模型的建立[J].中华肿瘤杂志,2004,26(8):461-464.
41.Elisabeth Z,Nancy W A,Mark D W,et al.Injection of human primary effusion lymphoma cells or associated macrophages into severe combined immunodeficient mice causes murine lymphomas[J].Cancer Res,2002,62:5536-5542.
42.Yanagisawa Y,Sato Y,Asahi-Ozaki Y,et al.Effusion and solid lymphomas have distinctive gene and protein expression profiles in an animal model of primary effusion lymphoma[J].J Pathol,2006,209(4):464-473.
43.Green PH,F leischauer A T,Bhagat G,et al.Risk of malignancy in patients with celiac disease[J].Am J M ed,2003,115(3):191-195.
44.Marafioti T,Pozzobon M,Hansmann ML,et al.Expression of intracellular signaling molecules in classical and lymphocyte predominance Hodgkin disease.Blood,2004;103(1):188-193.
45.Malec M,Soderqvist M,Sirsjo A,et al.Real-time polymerase chain reaction determination of cytokine mRNA expression profiles in Hodgkin's lymphoma.Haematologica.2004,89(6):679-85.
46.Karube K,Ohshima K,Suzumiya J,et al Gene expression profile of cytokines and chemokines in microdissected primary Hodgkin and Reed-Sternberg(H/RS) cells:high expression of interleukin- 11 receptor alpha.Ann Oncol.2006,17(1):110-6.
47.Poppema S,van den Berg A.Interaction between host T cells and Reed-Sternberg cells in Hodgkin lymphomas.Semin Cancer Biol.2000,10:345-350.
48.Aldinucci D,Lorenzon D,Olivo K,et al.Interactions between tissue fibroblasts in lymph nodes and Hodgkin/Reed-Sternberg cells.Leuk Lymphoma.2004,45(9):1731-9.
1.Br(a|¨)uninger A,Schmitz R,Bechtel D,et al.Molecular biology of Hodgkin's and Reed/Steinberg cells in Hodgkin's lymphoma[J],lnt J Cancer,2006,118(8):1853-61.
2.Kim SH,Shin YK,Lee IS,et al.Viral latent membrane protein 1(LMP-I) -induced CD99down-regulation in B cells leads to the generation of cells with Hodgkin's and Reed-Sternberg phenotype[J].Blood,2000,95(1):294-300.
3.沈丽佳,方唯意,谢思明,等小鼠B淋巴瘤A20细胞株mCD99L2基因表达检测及真核表达载体的构建[J].南方医科大学学报,2006,26(2):144-149.
4.何滢,沈丽佳,李祖国,等.mCD99L2基因沉默对小鼠B淋巴瘤细胞系A20细胞转化为H/RS样细胞的影响[J].基础医学与临床,2007,27(6):616-620.
5.K(u|¨)ppers R and Hansmann ML.The Hodgkin and Reed/Sternberg cell.IJBCB,2005;37(3):511-517.
6.Poppema S,van den Berg A.Interaction between host T cells and Reed-Sternberg cells in Hodgkin lymphomas[J].Semin Cancer Biol.2000;10:345-350.
7.K(u|¨)ppers R.B cells under influence:transformation orb cells by Epstein-Barr virus.Nat Rev Immunol,2003;3(10):801-12.
8.Zongli Qi,Tong Zhao,Xinhua Zhou,et al.EBER-1,ISH,LMP1 Immuno- histochemical tests and PCR for detecting Epstein-Barr Virus from Hodgkin's lymphoma tissues in southern China.U.S.Chinese Journal of Lymphology and Oncology,2004;3(1):5-10.
9.Kim SH,Shin YK,Lee IS,et al.Viral latent membrane protein 1(LMP-1)-induced CD99down-regulation in B cells leads to the generation of cells with Hodgkin's and Reed-Stembergphenotype[J].Blood,2000,95(1):294-300.
10.Imbert AM,Belaaloui G,Bardin F,et al.CD99 expressed on human mobilized peripheral blood CD34+ cells is involved in transendothelial migration.Blood,2006;108(8):2578-86.
11.Dworzak,MN,Froschl,G,Printz,D,et al.CD99 expression in T-lineage ALL:implications for flow cytometric detection of minimal residual disease.Leukemia,2004;18:703-708.
12.Manara MC,Bernard G,Lollini PL,et al.CD99 acts as an Oncosuppressor in osteosarcoma.Mol Biol Cell.2006;17(4):1910-1921.
13.Lee IS,Kim SH,Song HG,et al.The molecular basis for the generation of Hodgkin and Reed-Sternberg cells in Hodgkin's lymphoma,Int J Hematol.,2003;77(4):330-5.
14.Sub YH,Kim MK,Shin YK,et al.Mutations of the immunoglobulin heavy chain variable region gene in CD99-deficient BJAB cells line.Mol Cells,2002;13:237 -244.
15.李先茂,李燕,赵彤,等.霍奇金淋巴瘤CD99基因表达缺失的意义.第四军医大学学报,2004;25(23):2136-2137.
16.沈丽佳,何滢,蒋会勇,等.mic2/CD99在经典型霍奇金淋巴瘤H/RS细胞中的表达及与Eber-1/LMP-1相关性的研究.中国病理生理杂志,2006;22(4):776-780.
17.Suh YH,Shin YK,Kook MCH,et al.Cloning,genomic organization,alternative transcripts and expression analysis of CD99L2,a novel paralog of human CD99,and identification of evolutionary conserved motifs.Gene,2003,307:63-76.
18.Garbriele B,Stephan K,Stefan B,et al.Mouse CD99 participates in T-cell recruitment into inflamed skin.Blood.,2004;104:3205-3213.
19.Schenkel AR,Dufour EM,Chew TW,et al.The Murine CD99-Related Molecule CD99-Like 2(CD99L2) Is an Adhesion Molecule Involved in the Inflammatory Response.Cell Commun Adhes.2007;14(5):227-37.
20.沈丽佳,方唯意,谢思明,等小鼠B淋巴瘤A20细胞株mCD99L2基冈表达检测及真核表达载体的构建[J].南方医科大学学报,2006,26(2):144-149.
2].何滢,沈丽佳,李机国,等.mCD99L2基因沉默对小鼠B淋巴瘤细胞系A20细胞转化为H/RS样细胞的影响[J].基础医学与临床,2007,27(6):616-620.
22.Tzankov A,Zimpfer A,Went P,et al.Aberrant expression of cell cycle regulators in Hodgkin and Reed-Sternberg cells of classical Hodgkin's lymphoma.Mod Pathol.2005,18(1):90-6.
23.Leoncini L,Megha T,Lazzi S,et al.Cellular kinetic differences between Hodgkin's and anaplastic large cell lymphomas:relation to the expression of p34cdc2 and cyclin B-1.Int J Cancer.1998,77(3):408-14.
24.Gabriele B,Stephan K,Stefan B,et al.Mouse CD99 participates in T-cell recruitment into inflamed skin[J].Blood,2004,104(10):3205-13.
25.Bixel MG,Petri B,Khandoga AG,et al.A CD99-related antigen on endothelial cells mediates neutrophil,but not lymphocyte extravasation in vivo[J].Blood,2007,109(12):5327-36.
1.Kuppers R and Hansmann M L.The Hodgkin and Reed/Sternberg cell[J].IJBCB,2005,37(3):511-517.
2.Martin W,UIrike S,Andreas B,et al.3-[~(18)F]Fluoro-3'-Deoxythymidine([~(18)F]) as positron emission tomography tracer for imaging proliferation in a murine B-cell lymphoma model and in the human disease[J].Cancer Res,2003,63..2681-2687.
3.邵晓枫,杨纯正,熊冬生,等.人B淋巴瘤裸小鼠腹腔内移植模型[J].中国肿瘤临床,2001,28(3):217-219.
4.张宁,脱帅,刘秋珍.人肝恶性淋巴瘤裸小鼠原位及皮下移植模型的建立[J].消化外科,2006,5(1):57-61.
5.刘秋珍,脱朝伟,张宁等.人原发性直肠恶性淋巴瘤裸小鼠原位移植模型的建立及其生物学特性[J].消化外科,2006,5(3):206-210.
6.Elisabeth Z,Nancy W A,Mark D W,et al.Injection of human primary effusion lymphoma cells or associated macrophages into severe combined immunodeficient mice causes murine lymphomas[J].Cancer Res,2002,62:5536-5542.
7.Yanagisawa Y,Sato Y,Asahi-Ozaki Y,et al.Effusion and solid lymphomas have distinctive gene and protein expression profiles in an animal model of primary effusion lymphoma[J].J Pathol,2006,209(4):464-473.
8.Green PH,F leischauer A T,Bhagat G,et al.Risk of malignancy in patients with celiac disease[J].AmJMed,2003,115(3):191-195.
9.孙琦,陈辉树.恶性淋巴瘤的病因学研究进展.国际输血及血液学杂志[J],2006,29(5):398-402.
10.Hayashi K,Teramoto N,Akagi T.Animal in vivo models of EBV-associated lympho -proliferative disease:special references to rabbit models[J].Histo Histopathol,2002,17(4):1293-1310.
11.Burgess S C,Young J R,Baaten B J,et al.Marek's disease is a natural model for lymphomas over-expressing Hodgkin's disease antigen[J].PNAS,2004,101(38):13879-13884.
12.Osterrieder N,Kamil J P,Schumacher D,Tischer B K,Trapp S.Marek's disease virus:from miasmato model.Nat Rev Microbiol,2006,4(4):283-294.
13.Hasegawa H,Sawa H,Lewis M J,et al.Thymus-derived leukemia-lymphoma in mice transgenic for the Tax gene of human T-lymphotropic virus type Ⅰ.Nat Med,2006,12(4):466-472.
14.Nilsson J A,Nilsson L M,Keller U,et al.Id_2 is dispensable for myc-induced lymphoma genesis[J].Cancer Res,2004,64(20):7296-7301.
15.Beverly W B,John A,Anthony M,et al.The human BCL-6 transgene promotes the development of lymphomas in the mouse[J].PNAS,2004,101(39):14198-14203.
16.Brandt J VD,Kwon S H,McPherson K G,et al.Unexpected features of acute T lymphoblasti lymphomas in NotchlIC transgenic rats.EurJ Immunol,2006,36(8):2223-2234.
17.Martin W,Ulrike S,Andreas B,et al.3-[~(18)F]Fluoro-3'-Deoxythymidine([~(18)F]) as positron emission tomography tracer for imaging proliferation in a murine B-cell lymphoma model and in the human disease[J].Cancer Res,2003,63:2681- 2687.
18.邵晓枫,杨纯正,熊冬生,等.人B淋巴瘤裸小鼠腹腔内移植模型[J].中国肿瘤临床,2001,28(3):217-219.
19.刘秋珍,脱朝伟,张宁,等.人小肠原发性恶性淋巴瘤裸鼠原位移植模型的建立[J].中华肿瘤杂志,2004,26(8):461-464.
20.龚张斌,陈涛.胃癌动物模型制作概况[J].肿瘤学杂志,2002,8(5):35-37.
21.耿敬妹,薛英威,刘秀华,等.人消化道裸鼠移植瘤株及体外细胞系的建立[J].实用肿瘤学杂志,2000,14(3):162-164.
22.施新猷,王四旺,顾为望,等.比较医学.西安:陕西科学技术出版社,2003.478-480.
1.Oh KI,Kim BK,Ban YL,et al.CD99 activates T cells via a costimulatory function that promotes raft association of TCR complex and tyrosine phosphorylation of TCR zeta.Exp Mol Med.2007,39(2):176-84.
2.Choi EY,Park WS,Jung KC,et al.Engagement of CD99 induces up-regulation of TCR and MHC class Ⅰ and Ⅱ molecules on the surface of human thymocytes.J Immunol.1998,161(2):749-54.
3.Yoon SS,Kim H J,Chung DH,et al.CD99 costimulation up-regulatesT cell receptormediated activation of JNK and AP-l.Mol Cells.2004,18(2):186-91.
4. Pettersen RD, Bernard G, Olafsen MK, et al. CD99 signals caspase-independent T cell death.J Immunol. 2001, 166(8): 4931-42.
5. Suh YH, Shin YK, Kook MCH, et al. Cloning, genomic organization, alternative transcripts and expression analysis of CD99L2, a novel paralog of human CD99, and identification of evolutionary conserved motifs. Gene, 2003, 307: 63-76.
6. Gabriele B, Stephan K, Stefan B, et al. Mouse CD99 participates in T-cell recruitment into inflamed skin. Blood., 2004, 104: 3205-3213.
7. Schenkel AR, Dufour EM, Chew TW, et al. The murine CD99-related molecule CD99-Like 2 (CD99L2) is an adhesion molecule involved in the inflammatory response. Cell Commun Adhes. 2007, 14(5): 227-37.
8. van Wanrooij EJ, de Vos P, Bixel MG, et al.. Vaccination against CD99 inhibits athero genesis in low-density lipoprotein receptor-deficient mice. Cardiovasc Res. 2008 Mar 3 [Epub ahead of print].
9. Glaser SL, Clarke CA, Gulley ML, et al. Population-based patterns of human immunodeficiency virus-related Hodgkin lymphoma in the Greater San Francisco Bay Area,1988-1998. Cancer. 2003, 98: 300-309.
10. Hjalgrim H, Askling J, Rostgaard K, et al. Characteristics of Hodgkin lymphoma after infectious mononucleosis. N Engl J Med. 2003, 349: 1324-1332
11. Diepstra A, Boot M, van den Berg A, et al. HLA-G expression in classical Hodgkin lymphoma. Blood. 2003, 102: 897A-897A.
12. Diepstra A, Poppema S, Boot M, et al .HLA-G protein expression as a potential immune escape mechanism in classical Hodgkin's lymphoma. Tissue Antigens. 2008, 71(3): 219-26.
13. Poppema S, Porters M, Visser L, van den Berg AM. Immune escape mechanisms in Hodgkin's disease. Ann Oncol.1998;9 Suppl 5: S21-4.
14. Ohshima K, Muta K, Nakashima M, et al. Expression of human tumor-associated antigen RCAS1 in Reed-Stemberg cells in association with Epstein-Barr virus infection: a potential mechanism of immune evasion. Int J Cancer.2001, 93: 91-96.
15. Bladergroen BA, Meijer CJ, ten Berge RL, et al. Expression of the granzyme B inhibitor,protease inhibitor 9, by tumor cells in patients with non-Hodgkin and Hodgkin lymphoma: a novel protective mechanism for tumor cells to circumvent the immune system? Blood. 2002,99: 232-237.
16. Poppema S, van den Berg A. Interaction between host T cells and Reed-Sternberg cells in Hodgkin lymphomas.Semin Cancer Biol. 2000, 10: 345-350.
17. Malec M, Soderqvist M, Sirsjo A, et al .Real-time polymerase chain reaction determination of cytokine mRNA expression profiles in Hodgkin's lymphoma. Haematologica. 2004,89(6): 679-85.
18. Karube K, Ohshima K, Suzumiya J, et al Gene expression profile of cytokines and chemokines in microdissected primary Hodgkin and Reed-Sternberg (H/RS) cells: high expression of interleukin-11 receptor alpha. Ann Oncol. 2006, 17(1): 110-6.
19. Skinnider BF, Elia AJ, Gascoyne RD, et al. Interleukin 13 and interleukin 13 receptor are frequently expressed by Hodgkin and Reed-Sternberg cells of Hodgkin lymphoma. Blood.2001,97:250-255.
20. Terabe M, Park JM, Berzofsky JA. Role of IL-13 in regulation of anti-tumor immunity and tumor growth. Cancer Immunol Immunother. 2004, 53: 79-85.
1. Malec M, Soderqvist M, Sirsjo A, et al .Real-time polymerase chain reaction determination of cytokine mRNA expression profiles in Hodgkin's lymphoma[J]. Haematologica. 2004,89(6): 679-85.
2. Karube K, Ohshima K, Suzumiya J, et al Gene expression profile of cytokines and chemokines in microdissected primary Hodgkin and Reed-Sternberg (H/RS) cells: high expression of interleukin-11 receptor alpha[J]. Ann Oncol. 2006, 17(1):110-6.
3. Poppema S, van den Berg A. Interaction between host T cells and Reed-Sternberg cells in Hodgkin rymphomas[J].Semin Cancer Biol. 2000; 10(3): 345-350.
4. Aldinucci D, Lorenzon D, Olivo K, et al. Interactions between tissue fibroblasts in lymph nodes and Hodgkin/Reed-Sternberg cells[J]. Leuk Lymphoma. 2004, 45(9): 1731-9.
5. Hori R, Watanabe T, Ito K, et al. Cytoplasmic aggeegation of TRAF2 and TRAF5 proteins in the Hodgkin-Reed-Sternberg cells[J]. Am J Pathol.2002, 160: 1647-1654.
6. Horie R, Watanabe T, Morishita Y, et al. Ligand-independent signaling by overexpressed CD30 drives NF-kappaB activation in Hodgkin-Reed-Sternberg cells[J]. Oncogene. 2002,21(2): 2493-2503.
7. Gauld SB, Dal Porto JM, Cambier JC. B cell antigen receptor signaling: roles in cell development and disease [J]. Science,2002, 296 (5573): 1641-1642.
8. Zhu N, Ramirez LM, Lee RL, et al. CD40 signaling in B cells regulates the expression of the Pim21 kinase via the NF-κB pathway [J]. J Immunol, 2002, 168 (2): 744-754.
9. Hata K, Yoshimoto T, Mizuguchi J. CD40 ligand rescues inhibitor of differentiation-mediated Gl arrest induced by anti-IgM in WEH 12231 B lymphoma cells [J]. J Immunol,2004, 173(4): 2453-461.
10. Hollmann CA, Gong Q, Owens T. CD40-mediated apoptosis in murine B-lymphoma lines containing mutated p53 [J]. Exp Cell Res, 2002, 280 (2): 201-211.
11. Arimura Y, OgimotoM, Mitomo K, et al. CD45 is required for CD40-induced inhibition of DNA synthesis and regulation of c-Jun NH2-terminal kinase and p38 in BAL217 B cells[ J ]. J BioChem, 2001, 276 (11): 8550-8556.
12. Bevberich2Siebelf F, KussAW, KnEdelM, et al. A1 expression is stimulated by CD40 in B cells and rescues WEH I 231 cells from anti-IgM-induced cell death[ J ]. Eur J Immunol, 1999, 29(10): 3077-3088.
13. Craxton A, Chuang P I, Shu G, et al. The CD40-inducibie Bcl-family memberA1 protects B cells from antigen receptor—mediated apoptosis[J]. Cell Immunol, 2000, 200 (1): 56-62.
14. Lee IS, Kim SH, Song HG, et al .The molecular basis for the generation of Hodgkin and Reed-Sternberg cells in Hodgkin's lymphoma[J]. Int J Hematol, 2003, 77(4): 330-5
15 David A.Tthorley-Lawson, Andrew Gross Persistence of the Epstein-Barr Virus and the Origins of Associted Lymphomas[J]. N Engl J Med. 2004, 350: 1328-1337
16. Aldinucci D, Olivo K, Lorenzon D, et al. The role of interleukin-3 in classical Hodgkin's disease [J]. Leuk Lymphoma. 2005, 46(3): 303-11.
17. Niederkorn JY.Emerging concepts in CD8(+) T regulatory cells[J].Curr Opin Immunol.2008 Apr 10 [Epub ahead of print]
18. Wuttge DM, Zhou X, Sheikine Y, et al. CXCL16/SR-PSOX is an interferon-gamma-regulated chemokine and scavenger receptor expressed in atherosclerotic lesions[J].Arterioscler Thromb Vasc Biol. 2004, 24(4): 750-5.
19. Luft T, Luetjens P, Hochrein H, et al. IFN-alpha enhances CD40 ligand-mediated activation of immature monocyte-derived dendritic cells[J]. Int Immunol. 2002, 14(4): 367-80.
20. Suzuki K, Nakaji S, Kurakake S, et al. Exhaustive exercise and type-1/type-2 cytokine balance with special focus on interleukin-12 p40/p70[J]. Exerc Immunol Rev. 2003, 9:48-57.
21. Aldinucci D, Lorenzon D, Cattaruzza L, et al. Expression of CCR5 receptors on Reed-Sternberg cells and Hodgkin lymphoma cell lines: involvement of CCL5/Rantes in tumor cell growth and microenvironmental interactions[J]. Int J Cancer. 2008, 122(4): 769-76.
22. Commins S, Steinke JW, Borish L。 The extended IL-10 superfamily: IL-10, IL-19, IL-20,IL-22, IL-24, IL-26, IL-28, and IL-29[J]. J Allergy Clin Immunol. 2008 Apr 9 [Epub ahead of print]
23. Maggio E, van den Berg A, Diepstra A, et al .Chemokines, cytokines and their receptors in Hodgkin's lymphoma cell lines and tissues[J]. Ann Oncol. 2002, 13 Suppl 1: 52-6.
24. Marshall NA, Christie LF, Munro LR, et al. Immunosuppressive regulatory T cells are abundant in the reactive lymphocytes of Hodgkin lymphoma[J]. Blood. 2004, 103: 1755-1762.
25. Samanta M, Iwakiri D, Takada K.Epstein-Barr virus-encoded small RNA induces IL-10 through RIG-I-mediated IRF-3 signaling[J]. Oncogene. 2008 Mar 24 [Epub ahead of print]
26. Irons RD, Le AT.Dithiocarbamates and viral IL-10 collaborate in the immortalization and evasion of immune response in EBV-infected human B Iymphocytes[J]. Chem Biol Interact.2008, 172(1): 81-92.
27. da Silva GN, Bacchi MM, Rainho CA, et al.Epstein-Barr virus infection and single nucleotide polymorphisms in the promoter region of interleukin 10 gene in patients with Hodgkin lymphoma[J]. Arch Pathol Lab Med. 2007, 131 (11): 1691 -6.
28. Marshall NA, Culligan DJ, Tighe J, et al.The relationships between Epstein-Barr virus latent membrane protein 1 and regulatory T cells in Hodgkin's lymphoma[J]. Exp Hematol. 2007,35(4): 596-604.
29. 路慧丽,俞眉,韩伟.趋化因子CXCL9/Mig的研究进展中国生物工程杂志, 2006,26(10):59-63.
30. Yao TC , Kuo ML , See LC , et al . The RANTES promoter polymorphism: a genetic risk factor for near-fatal asthma in Chinese children[J] . J Allergy Clin Immunol,2003,l 11 (6):1285- 1292.
31. Muller K, Bischof S, Sommer F, et al.Differential production of macrophage inflammatory protein 1 gamma (MIP-lgamma), lymphotactin, and MIP-2 by CD4(+) Th subsets polarized in vitro and in vivo[J]. Infect Immun. 2003, 71(11): 6178-83.
32. Reynolds GM, Billingham LJ, Gray LJ, et al. Interleukin 6 expression by Hodgkin/Reed-Sternberg cells is associated with the presence of 'B' symptoms and failure to achieve complete remission in patients with advanced Hodgkin's disease[J]. Br J Haematol. 2002,118(1): 195-201.
33. Kobayashi K, Yokote T, Akioka T, et al. Vascular endothelial growth factor and interleukin 6 production by Hodgkin lymphoma[J]. Gan To Kagaku Ryoho. 2007, 34(8): 1327-30.
34. Smyth DC, Kerr C, Li Y, et al. Oncostatin M induction of eotaxin-1 expression requires the convergence of PI3'K and ERK1/2 MAPK signal transduction pathways[J]. Cell Signal.2008 Feb 11 [Epub ahead of print]
35. Mukherjee A, Rotwein P. Insulin-like growth factor binding protein-5 in osteogenesis:facilitator or inhibitor? Growth Horm IGF Res. 2007, 17(3): 179-85.
36.Strohbach C,Kleinman S,Linkhart T,et al.Potential involvement of the interaction between insulin-like growth factor binding protein(1GFBP)-6 and LIM mineralization protein (LMP)-I in regulating osteoblast differentiation[J].J Cell Biochem.2008 Apr 7[Epub ahead of print]
37.Pazaitou-Panayiotou K,Kelesidis T,Kelesidis I,et al.Growth hormone-binding protein is directly and IGFBP-3 is inversely associated with risk of female breast cancer[J].Eur J Endocrinol.2007,156(2):187-94.
38.Fu P,Thompson JA,Bach LA.Promotion of cancer cell migration:an insulin-like growth factor(IGF)-independent action of IGF-binding protein-6[J].J Biol Chem.2007,282(31):22298-306.
39.Shimaoka T,Seino K,Kume N,et al.Critical role for CXC chemokine ligand 16(SR-PSOX)in Th1 response mediated by NKT cells[J].J Immunol.2007,179(12):8172-9.
40.Kobayashi K,Yokote T,Akioka T,et al.Vascular endothelial growth factor and interleukin 6 production by Hodgkin lymphoma[J].Gan To Kagaku Ryoho.2007,34(8):1327-30.
2. Kuppers R. B cells under influence: transformation of B cells by Epstein-Barr virus. Nat Rev Immunol, 2003,3(10):801-12.
3. Zongli Qi, Tong Zhao, Xinhua Zhou, et al. EBER-1, ISH, LMP1 Immuno- histochemical tests and PCR for detecting Epstein-Barr Virus from Hodgkin's lymphoma tissues in southern China. U.S.Chinese Journal of Lymphology and Oncology, 2004,3 (1):5-10.
4. Kang LC, Dunphy CH.Immunoreactivity of M1C2 (CD99) and terminal deoxynucleotidyl transferase in bone marrow clot and core specimens of acute myeloid leukemias and myelodysplastic syndromes. Arch Pathol Lab Med. 2006, 130(2): 153-7.
5. Dworzak MN, Fritsch G, Fleischer C, et al .CD99 (MIC2) expression in paediatric B-lineage leukaemia/lymphoma reflects maturation- associated patterns of normal B-lymphopoiesis. Br J Haematol. 1999, 105(3):690-5.
6. Hystad ME, Myklebust JH, Bo TH, et al .Characterization of early stages of human B cell development by gene expression profiling. J Immunol. 2007, 179 (6):3662-71.
7. Miyagawa Y, Okita H, Nakaijima H, et al.Inducible expression of chimeric EWS/ETS proteins confers Ewing's family tumor-like phenotypes to human mesenchymal progenitor cells. Mol Cell Biol. 2008 Jan 22 [Epub ahead of print]
8. Dworzak, MN, Froschl, G, Printz, D, et al. CD99 expression in T-lineage ALL: implications for flow cytometric detection of minimal residual disease [J]. Leukemia, 2004, 18(4):703-8.
9. Shapiro M, Wasik MA, Junkins-Hopkins JM, et al. Complete remission in advanced blastic NK-cell lymphoma/leukemia in elderly patients using the hyper-CVAD regimen. Am J Hematol.2003, 74(1):46-51.
10. Odashiro AN, Leite LV, Oliveira RS, et al. Primary orbital mesenchymal chondrosarcoma:a case report and literature review. Int Ophthalmol. 2008 Jan 10 [Epub ahead of print]
11. Diwan AH, Skelton HG 3rd, Horenstein MG, et al.Dermatofibrosarcoma protuberans and giant cell fibroblastoma exhibit CD99 positivity. J Cutan Pathol. 2008 Jan 14 [Epub ahead of print]
12.Ramsay AD,Bates AW,Williams S,et al.Variable antigen expression in hepatoblastomas.Appl Immunohistochem Mol Morphol.2008 Jan 25[Epub ahead of print]
13.Park CK,Shin YK,Kim TJ,et aI.High CD99 expression in memory T and B cells in reactive lymph nodes.J Korean Med Sci.1999,14(6):600-6.
14.Manara MC,Bernard G,Lollini PL,et al.CD99 acts as an oncosuppressor in osteosarcoma [J].Mol Biol Cell,2006,17(4):1910-21.
15.Imbert AM,Belaaloui G,Bardin F,et ai.CD99 expressed on human mobilized peripheral blood CD34+ cells is involved in trans- endothelial migration[J].Blood,2006,108(8):2578-86.
16.Zucchini C,Rocchi A,Manara MC,et al.Apoptotic genes as potential markers of metastatic phenotype in human osteosarcoma cell lines.Int J Oncol.2008,32(1):17-31.
17.Lee JH,Kim SH,Wang LH,et al.Clinical significance of CD99 down- regulation in gastric adenocarcinoma.Clin Cancer Res.2007,13(9):2584-91.
18.Scotlandi K,Zuntini M,Manara MC,et al.CD99 isoforms dictate opposite functions in tumour malignancy and metastases by activating or repressing c-Src kinase activity.Oncogene.2007,26(46):6604-18.
19.Byun H J,Hong IK,Kim E,et ai.A splice variant of CD99 increases motility and MMP-9expression of human breast cancer cells through the AKT-,ERK-,and JNK-dependent AP-1 activation signaling pathways.J Biol Chem.2006,281(46):34833-47.
20.Lee IS,Kim SH,Song HG,et al.The molecular basis for the generation of Hodgkin and Reed-Sternberg cells in Hodgkin's iymphoma.Int J Hematol.,2003,77(4):330-5.
21.Sub YH,Kim MK,Shin YK,et al.Mutations of the immunoglobulin heavy chain variable region gene in CD99-deficient B JAB cells line.Mol Cells,2002,13:237 -244.
22.李先茂,李燕,赵彤,等.霍奇金淋巴瘤CD99基因表达缺失的意义.第四军医大学学报,2004.25(23):2136-2137.
23.沈丽佳,何滢,蒋会勇,等.mic2/CD99在经典型霍奇金淋巴瘤H/RS细胞中的表达及与Eber-1/LMP-1相关性的研究.中国病理生理杂志,2006,22(4):776-780.
24.何滢,硕士论文.L428细胞CD99基因上调H/RS细胞形态转化及免疫表型的研究.
25.Suh YH,Shin YK,Kook MCH,et al.Cloning,genomic organization,alternative transcripts and expression analysis of CD99L2,a novel paralog of human CD99,and identification of evolutionary conserved motifs.Gene,2003,307:63-76.
26.Gabriele B,Stephan K,Stefan B,et al.Mouse CD99 participates in T-cell recruitment into inflamed skin.Blood.,2004,104:3205-3213.
27.Schenkel AR,Dufour EM,Chew TW,et al.The murine CD99-related molecule CD99-1ike 2(CD99L2) is an adhesion molecule involved in the inflammatory response.Cell Commun Adhes.2007,14(5):227-37.
28.van Wanrooij EJ,de Vos P,Bixel MG,et al..Vaccination against CD99 inhibits athero genesis in low-density lipoprotein receptor-deficient mice.Cardiovasc Res.2008 Mar 3[Epub ahead of print]
29.沈丽佳,方唯意,谢思明,等.小鼠B淋巴瘤A20细胞株mCD99L2基冈表达检测及真核表达载体的构建.南方医科大学学报,2006,26(2):144-149.
30.何滢,沈丽佳,李祖国,等.mCD99L2基因沉默对小鼠B淋巴瘤细胞系A20细胞转化为H/RS样细胞的影响.基础医学与临床,2007,27(6):616-620.
31.Hayashi K,Teramoto N,Akagi T.Animal in vivo models of EBV-associated lympho -proliferative disease:special references to rabbit models[J].Histo Histopathol,2002,17(4):1293-1310.
32.Burgess S C,Young J R,Baaten B J,et al.Marek's disease is a natural model for lymphomas over-expressing Hodgkin's disease antigen[J].PNAS,2004,101(38)..13879-13884.
33.Osterrieder N,Kamil J P,Schumacher D,Tischer B K,Trapp S.Marek's disease virus:from miasma to model.Nat Rev Microbiol,2006,4(4).- 283-294.
34.Hasegawa H,Sawa H,Lewis M J,et al.Thymus-derived leukemia-lymphoma in mice transgenic for the Tax gene of human T-lymphotropic virus type Ⅰ.Nat Med,2006,12(4):466-472.
35.Nilsson J A,Nilsson L M,Keller U,et al.Id_2 is dispensable for myc-induced lymphoma genesis[J].Cancer Res,2004,64(20):7296-7301.
36.Beverly W B,John A,Anthony M,et al.The human BCL-6 transgene promotes the development of lymphomas in the mouse[J].PNAS,2004,101(39):14198-14203.
37.Brandt J VD,Kwon S H,McPherson K G,et al.Unexpected features of acute T lymphoblastic lymphomas in NotchlIC transgenic rats.Eur J Immunol,2006,36(8):2223-2234.
38.Martin W,Ulrike S,Andreas B,et al.3-[~(18)F]Fluoro-3'-Deoxythymidine([~(18)F]) as positron emission tomography tracer for imaging proliferation in a murine B-cell lymphoma model and in the human disease[J].Cancer Res,2003,63:2681-2687.
39.邵晓枫,杨纯正,熊冬生,等.人B淋巴瘤裸小鼠腹腔内移植模型[J].中国肿瘤临床,2001,28(3):217-219.
40.刘秋珍,脱朝伟,张宁,等.人小肠原发性恶性淋巴瘤裸鼠原位移植模型的建立[J].中华肿瘤杂志,2004,26(8):461-464.
41.Elisabeth Z,Nancy W A,Mark D W,et al.Injection of human primary effusion lymphoma cells or associated macrophages into severe combined immunodeficient mice causes murine lymphomas[J].Cancer Res,2002,62:5536-5542.
42.Yanagisawa Y,Sato Y,Asahi-Ozaki Y,et al.Effusion and solid lymphomas have distinctive gene and protein expression profiles in an animal model of primary effusion lymphoma[J].J Pathol,2006,209(4):464-473.
43.Green PH,F leischauer A T,Bhagat G,et al.Risk of malignancy in patients with celiac disease[J].Am J M ed,2003,115(3):191-195.
44.Marafioti T,Pozzobon M,Hansmann ML,et al.Expression of intracellular signaling molecules in classical and lymphocyte predominance Hodgkin disease.Blood,2004;103(1):188-193.
45.Malec M,Soderqvist M,Sirsjo A,et al.Real-time polymerase chain reaction determination of cytokine mRNA expression profiles in Hodgkin's lymphoma.Haematologica.2004,89(6):679-85.
46.Karube K,Ohshima K,Suzumiya J,et al Gene expression profile of cytokines and chemokines in microdissected primary Hodgkin and Reed-Sternberg(H/RS) cells:high expression of interleukin- 11 receptor alpha.Ann Oncol.2006,17(1):110-6.
47.Poppema S,van den Berg A.Interaction between host T cells and Reed-Sternberg cells in Hodgkin lymphomas.Semin Cancer Biol.2000,10:345-350.
48.Aldinucci D,Lorenzon D,Olivo K,et al.Interactions between tissue fibroblasts in lymph nodes and Hodgkin/Reed-Sternberg cells.Leuk Lymphoma.2004,45(9):1731-9.
1.Br(a|¨)uninger A,Schmitz R,Bechtel D,et al.Molecular biology of Hodgkin's and Reed/Steinberg cells in Hodgkin's lymphoma[J],lnt J Cancer,2006,118(8):1853-61.
2.Kim SH,Shin YK,Lee IS,et al.Viral latent membrane protein 1(LMP-I) -induced CD99down-regulation in B cells leads to the generation of cells with Hodgkin's and Reed-Sternberg phenotype[J].Blood,2000,95(1):294-300.
3.沈丽佳,方唯意,谢思明,等小鼠B淋巴瘤A20细胞株mCD99L2基因表达检测及真核表达载体的构建[J].南方医科大学学报,2006,26(2):144-149.
4.何滢,沈丽佳,李祖国,等.mCD99L2基因沉默对小鼠B淋巴瘤细胞系A20细胞转化为H/RS样细胞的影响[J].基础医学与临床,2007,27(6):616-620.
5.K(u|¨)ppers R and Hansmann ML.The Hodgkin and Reed/Sternberg cell.IJBCB,2005;37(3):511-517.
6.Poppema S,van den Berg A.Interaction between host T cells and Reed-Sternberg cells in Hodgkin lymphomas[J].Semin Cancer Biol.2000;10:345-350.
7.K(u|¨)ppers R.B cells under influence:transformation orb cells by Epstein-Barr virus.Nat Rev Immunol,2003;3(10):801-12.
8.Zongli Qi,Tong Zhao,Xinhua Zhou,et al.EBER-1,ISH,LMP1 Immuno- histochemical tests and PCR for detecting Epstein-Barr Virus from Hodgkin's lymphoma tissues in southern China.U.S.Chinese Journal of Lymphology and Oncology,2004;3(1):5-10.
9.Kim SH,Shin YK,Lee IS,et al.Viral latent membrane protein 1(LMP-1)-induced CD99down-regulation in B cells leads to the generation of cells with Hodgkin's and Reed-Stembergphenotype[J].Blood,2000,95(1):294-300.
10.Imbert AM,Belaaloui G,Bardin F,et al.CD99 expressed on human mobilized peripheral blood CD34+ cells is involved in transendothelial migration.Blood,2006;108(8):2578-86.
11.Dworzak,MN,Froschl,G,Printz,D,et al.CD99 expression in T-lineage ALL:implications for flow cytometric detection of minimal residual disease.Leukemia,2004;18:703-708.
12.Manara MC,Bernard G,Lollini PL,et al.CD99 acts as an Oncosuppressor in osteosarcoma.Mol Biol Cell.2006;17(4):1910-1921.
13.Lee IS,Kim SH,Song HG,et al.The molecular basis for the generation of Hodgkin and Reed-Sternberg cells in Hodgkin's lymphoma,Int J Hematol.,2003;77(4):330-5.
14.Sub YH,Kim MK,Shin YK,et al.Mutations of the immunoglobulin heavy chain variable region gene in CD99-deficient BJAB cells line.Mol Cells,2002;13:237 -244.
15.李先茂,李燕,赵彤,等.霍奇金淋巴瘤CD99基因表达缺失的意义.第四军医大学学报,2004;25(23):2136-2137.
16.沈丽佳,何滢,蒋会勇,等.mic2/CD99在经典型霍奇金淋巴瘤H/RS细胞中的表达及与Eber-1/LMP-1相关性的研究.中国病理生理杂志,2006;22(4):776-780.
17.Suh YH,Shin YK,Kook MCH,et al.Cloning,genomic organization,alternative transcripts and expression analysis of CD99L2,a novel paralog of human CD99,and identification of evolutionary conserved motifs.Gene,2003,307:63-76.
18.Garbriele B,Stephan K,Stefan B,et al.Mouse CD99 participates in T-cell recruitment into inflamed skin.Blood.,2004;104:3205-3213.
19.Schenkel AR,Dufour EM,Chew TW,et al.The Murine CD99-Related Molecule CD99-Like 2(CD99L2) Is an Adhesion Molecule Involved in the Inflammatory Response.Cell Commun Adhes.2007;14(5):227-37.
20.沈丽佳,方唯意,谢思明,等小鼠B淋巴瘤A20细胞株mCD99L2基冈表达检测及真核表达载体的构建[J].南方医科大学学报,2006,26(2):144-149.
2].何滢,沈丽佳,李机国,等.mCD99L2基因沉默对小鼠B淋巴瘤细胞系A20细胞转化为H/RS样细胞的影响[J].基础医学与临床,2007,27(6):616-620.
22.Tzankov A,Zimpfer A,Went P,et al.Aberrant expression of cell cycle regulators in Hodgkin and Reed-Sternberg cells of classical Hodgkin's lymphoma.Mod Pathol.2005,18(1):90-6.
23.Leoncini L,Megha T,Lazzi S,et al.Cellular kinetic differences between Hodgkin's and anaplastic large cell lymphomas:relation to the expression of p34cdc2 and cyclin B-1.Int J Cancer.1998,77(3):408-14.
24.Gabriele B,Stephan K,Stefan B,et al.Mouse CD99 participates in T-cell recruitment into inflamed skin[J].Blood,2004,104(10):3205-13.
25.Bixel MG,Petri B,Khandoga AG,et al.A CD99-related antigen on endothelial cells mediates neutrophil,but not lymphocyte extravasation in vivo[J].Blood,2007,109(12):5327-36.
1.Kuppers R and Hansmann M L.The Hodgkin and Reed/Sternberg cell[J].IJBCB,2005,37(3):511-517.
2.Martin W,UIrike S,Andreas B,et al.3-[~(18)F]Fluoro-3'-Deoxythymidine([~(18)F]) as positron emission tomography tracer for imaging proliferation in a murine B-cell lymphoma model and in the human disease[J].Cancer Res,2003,63..2681-2687.
3.邵晓枫,杨纯正,熊冬生,等.人B淋巴瘤裸小鼠腹腔内移植模型[J].中国肿瘤临床,2001,28(3):217-219.
4.张宁,脱帅,刘秋珍.人肝恶性淋巴瘤裸小鼠原位及皮下移植模型的建立[J].消化外科,2006,5(1):57-61.
5.刘秋珍,脱朝伟,张宁等.人原发性直肠恶性淋巴瘤裸小鼠原位移植模型的建立及其生物学特性[J].消化外科,2006,5(3):206-210.
6.Elisabeth Z,Nancy W A,Mark D W,et al.Injection of human primary effusion lymphoma cells or associated macrophages into severe combined immunodeficient mice causes murine lymphomas[J].Cancer Res,2002,62:5536-5542.
7.Yanagisawa Y,Sato Y,Asahi-Ozaki Y,et al.Effusion and solid lymphomas have distinctive gene and protein expression profiles in an animal model of primary effusion lymphoma[J].J Pathol,2006,209(4):464-473.
8.Green PH,F leischauer A T,Bhagat G,et al.Risk of malignancy in patients with celiac disease[J].AmJMed,2003,115(3):191-195.
9.孙琦,陈辉树.恶性淋巴瘤的病因学研究进展.国际输血及血液学杂志[J],2006,29(5):398-402.
10.Hayashi K,Teramoto N,Akagi T.Animal in vivo models of EBV-associated lympho -proliferative disease:special references to rabbit models[J].Histo Histopathol,2002,17(4):1293-1310.
11.Burgess S C,Young J R,Baaten B J,et al.Marek's disease is a natural model for lymphomas over-expressing Hodgkin's disease antigen[J].PNAS,2004,101(38):13879-13884.
12.Osterrieder N,Kamil J P,Schumacher D,Tischer B K,Trapp S.Marek's disease virus:from miasmato model.Nat Rev Microbiol,2006,4(4):283-294.
13.Hasegawa H,Sawa H,Lewis M J,et al.Thymus-derived leukemia-lymphoma in mice transgenic for the Tax gene of human T-lymphotropic virus type Ⅰ.Nat Med,2006,12(4):466-472.
14.Nilsson J A,Nilsson L M,Keller U,et al.Id_2 is dispensable for myc-induced lymphoma genesis[J].Cancer Res,2004,64(20):7296-7301.
15.Beverly W B,John A,Anthony M,et al.The human BCL-6 transgene promotes the development of lymphomas in the mouse[J].PNAS,2004,101(39):14198-14203.
16.Brandt J VD,Kwon S H,McPherson K G,et al.Unexpected features of acute T lymphoblasti lymphomas in NotchlIC transgenic rats.EurJ Immunol,2006,36(8):2223-2234.
17.Martin W,Ulrike S,Andreas B,et al.3-[~(18)F]Fluoro-3'-Deoxythymidine([~(18)F]) as positron emission tomography tracer for imaging proliferation in a murine B-cell lymphoma model and in the human disease[J].Cancer Res,2003,63:2681- 2687.
18.邵晓枫,杨纯正,熊冬生,等.人B淋巴瘤裸小鼠腹腔内移植模型[J].中国肿瘤临床,2001,28(3):217-219.
19.刘秋珍,脱朝伟,张宁,等.人小肠原发性恶性淋巴瘤裸鼠原位移植模型的建立[J].中华肿瘤杂志,2004,26(8):461-464.
20.龚张斌,陈涛.胃癌动物模型制作概况[J].肿瘤学杂志,2002,8(5):35-37.
21.耿敬妹,薛英威,刘秀华,等.人消化道裸鼠移植瘤株及体外细胞系的建立[J].实用肿瘤学杂志,2000,14(3):162-164.
22.施新猷,王四旺,顾为望,等.比较医学.西安:陕西科学技术出版社,2003.478-480.
1.Oh KI,Kim BK,Ban YL,et al.CD99 activates T cells via a costimulatory function that promotes raft association of TCR complex and tyrosine phosphorylation of TCR zeta.Exp Mol Med.2007,39(2):176-84.
2.Choi EY,Park WS,Jung KC,et al.Engagement of CD99 induces up-regulation of TCR and MHC class Ⅰ and Ⅱ molecules on the surface of human thymocytes.J Immunol.1998,161(2):749-54.
3.Yoon SS,Kim H J,Chung DH,et al.CD99 costimulation up-regulatesT cell receptormediated activation of JNK and AP-l.Mol Cells.2004,18(2):186-91.
4. Pettersen RD, Bernard G, Olafsen MK, et al. CD99 signals caspase-independent T cell death.J Immunol. 2001, 166(8): 4931-42.
5. Suh YH, Shin YK, Kook MCH, et al. Cloning, genomic organization, alternative transcripts and expression analysis of CD99L2, a novel paralog of human CD99, and identification of evolutionary conserved motifs. Gene, 2003, 307: 63-76.
6. Gabriele B, Stephan K, Stefan B, et al. Mouse CD99 participates in T-cell recruitment into inflamed skin. Blood., 2004, 104: 3205-3213.
7. Schenkel AR, Dufour EM, Chew TW, et al. The murine CD99-related molecule CD99-Like 2 (CD99L2) is an adhesion molecule involved in the inflammatory response. Cell Commun Adhes. 2007, 14(5): 227-37.
8. van Wanrooij EJ, de Vos P, Bixel MG, et al.. Vaccination against CD99 inhibits athero genesis in low-density lipoprotein receptor-deficient mice. Cardiovasc Res. 2008 Mar 3 [Epub ahead of print].
9. Glaser SL, Clarke CA, Gulley ML, et al. Population-based patterns of human immunodeficiency virus-related Hodgkin lymphoma in the Greater San Francisco Bay Area,1988-1998. Cancer. 2003, 98: 300-309.
10. Hjalgrim H, Askling J, Rostgaard K, et al. Characteristics of Hodgkin lymphoma after infectious mononucleosis. N Engl J Med. 2003, 349: 1324-1332
11. Diepstra A, Boot M, van den Berg A, et al. HLA-G expression in classical Hodgkin lymphoma. Blood. 2003, 102: 897A-897A.
12. Diepstra A, Poppema S, Boot M, et al .HLA-G protein expression as a potential immune escape mechanism in classical Hodgkin's lymphoma. Tissue Antigens. 2008, 71(3): 219-26.
13. Poppema S, Porters M, Visser L, van den Berg AM. Immune escape mechanisms in Hodgkin's disease. Ann Oncol.1998;9 Suppl 5: S21-4.
14. Ohshima K, Muta K, Nakashima M, et al. Expression of human tumor-associated antigen RCAS1 in Reed-Stemberg cells in association with Epstein-Barr virus infection: a potential mechanism of immune evasion. Int J Cancer.2001, 93: 91-96.
15. Bladergroen BA, Meijer CJ, ten Berge RL, et al. Expression of the granzyme B inhibitor,protease inhibitor 9, by tumor cells in patients with non-Hodgkin and Hodgkin lymphoma: a novel protective mechanism for tumor cells to circumvent the immune system? Blood. 2002,99: 232-237.
16. Poppema S, van den Berg A. Interaction between host T cells and Reed-Sternberg cells in Hodgkin lymphomas.Semin Cancer Biol. 2000, 10: 345-350.
17. Malec M, Soderqvist M, Sirsjo A, et al .Real-time polymerase chain reaction determination of cytokine mRNA expression profiles in Hodgkin's lymphoma. Haematologica. 2004,89(6): 679-85.
18. Karube K, Ohshima K, Suzumiya J, et al Gene expression profile of cytokines and chemokines in microdissected primary Hodgkin and Reed-Sternberg (H/RS) cells: high expression of interleukin-11 receptor alpha. Ann Oncol. 2006, 17(1): 110-6.
19. Skinnider BF, Elia AJ, Gascoyne RD, et al. Interleukin 13 and interleukin 13 receptor are frequently expressed by Hodgkin and Reed-Sternberg cells of Hodgkin lymphoma. Blood.2001,97:250-255.
20. Terabe M, Park JM, Berzofsky JA. Role of IL-13 in regulation of anti-tumor immunity and tumor growth. Cancer Immunol Immunother. 2004, 53: 79-85.
1. Malec M, Soderqvist M, Sirsjo A, et al .Real-time polymerase chain reaction determination of cytokine mRNA expression profiles in Hodgkin's lymphoma[J]. Haematologica. 2004,89(6): 679-85.
2. Karube K, Ohshima K, Suzumiya J, et al Gene expression profile of cytokines and chemokines in microdissected primary Hodgkin and Reed-Sternberg (H/RS) cells: high expression of interleukin-11 receptor alpha[J]. Ann Oncol. 2006, 17(1):110-6.
3. Poppema S, van den Berg A. Interaction between host T cells and Reed-Sternberg cells in Hodgkin rymphomas[J].Semin Cancer Biol. 2000; 10(3): 345-350.
4. Aldinucci D, Lorenzon D, Olivo K, et al. Interactions between tissue fibroblasts in lymph nodes and Hodgkin/Reed-Sternberg cells[J]. Leuk Lymphoma. 2004, 45(9): 1731-9.
5. Hori R, Watanabe T, Ito K, et al. Cytoplasmic aggeegation of TRAF2 and TRAF5 proteins in the Hodgkin-Reed-Sternberg cells[J]. Am J Pathol.2002, 160: 1647-1654.
6. Horie R, Watanabe T, Morishita Y, et al. Ligand-independent signaling by overexpressed CD30 drives NF-kappaB activation in Hodgkin-Reed-Sternberg cells[J]. Oncogene. 2002,21(2): 2493-2503.
7. Gauld SB, Dal Porto JM, Cambier JC. B cell antigen receptor signaling: roles in cell development and disease [J]. Science,2002, 296 (5573): 1641-1642.
8. Zhu N, Ramirez LM, Lee RL, et al. CD40 signaling in B cells regulates the expression of the Pim21 kinase via the NF-κB pathway [J]. J Immunol, 2002, 168 (2): 744-754.
9. Hata K, Yoshimoto T, Mizuguchi J. CD40 ligand rescues inhibitor of differentiation-mediated Gl arrest induced by anti-IgM in WEH 12231 B lymphoma cells [J]. J Immunol,2004, 173(4): 2453-461.
10. Hollmann CA, Gong Q, Owens T. CD40-mediated apoptosis in murine B-lymphoma lines containing mutated p53 [J]. Exp Cell Res, 2002, 280 (2): 201-211.
11. Arimura Y, OgimotoM, Mitomo K, et al. CD45 is required for CD40-induced inhibition of DNA synthesis and regulation of c-Jun NH2-terminal kinase and p38 in BAL217 B cells[ J ]. J BioChem, 2001, 276 (11): 8550-8556.
12. Bevberich2Siebelf F, KussAW, KnEdelM, et al. A1 expression is stimulated by CD40 in B cells and rescues WEH I 231 cells from anti-IgM-induced cell death[ J ]. Eur J Immunol, 1999, 29(10): 3077-3088.
13. Craxton A, Chuang P I, Shu G, et al. The CD40-inducibie Bcl-family memberA1 protects B cells from antigen receptor—mediated apoptosis[J]. Cell Immunol, 2000, 200 (1): 56-62.
14. Lee IS, Kim SH, Song HG, et al .The molecular basis for the generation of Hodgkin and Reed-Sternberg cells in Hodgkin's lymphoma[J]. Int J Hematol, 2003, 77(4): 330-5
15 David A.Tthorley-Lawson, Andrew Gross Persistence of the Epstein-Barr Virus and the Origins of Associted Lymphomas[J]. N Engl J Med. 2004, 350: 1328-1337
16. Aldinucci D, Olivo K, Lorenzon D, et al. The role of interleukin-3 in classical Hodgkin's disease [J]. Leuk Lymphoma. 2005, 46(3): 303-11.
17. Niederkorn JY.Emerging concepts in CD8(+) T regulatory cells[J].Curr Opin Immunol.2008 Apr 10 [Epub ahead of print]
18. Wuttge DM, Zhou X, Sheikine Y, et al. CXCL16/SR-PSOX is an interferon-gamma-regulated chemokine and scavenger receptor expressed in atherosclerotic lesions[J].Arterioscler Thromb Vasc Biol. 2004, 24(4): 750-5.
19. Luft T, Luetjens P, Hochrein H, et al. IFN-alpha enhances CD40 ligand-mediated activation of immature monocyte-derived dendritic cells[J]. Int Immunol. 2002, 14(4): 367-80.
20. Suzuki K, Nakaji S, Kurakake S, et al. Exhaustive exercise and type-1/type-2 cytokine balance with special focus on interleukin-12 p40/p70[J]. Exerc Immunol Rev. 2003, 9:48-57.
21. Aldinucci D, Lorenzon D, Cattaruzza L, et al. Expression of CCR5 receptors on Reed-Sternberg cells and Hodgkin lymphoma cell lines: involvement of CCL5/Rantes in tumor cell growth and microenvironmental interactions[J]. Int J Cancer. 2008, 122(4): 769-76.
22. Commins S, Steinke JW, Borish L。 The extended IL-10 superfamily: IL-10, IL-19, IL-20,IL-22, IL-24, IL-26, IL-28, and IL-29[J]. J Allergy Clin Immunol. 2008 Apr 9 [Epub ahead of print]
23. Maggio E, van den Berg A, Diepstra A, et al .Chemokines, cytokines and their receptors in Hodgkin's lymphoma cell lines and tissues[J]. Ann Oncol. 2002, 13 Suppl 1: 52-6.
24. Marshall NA, Christie LF, Munro LR, et al. Immunosuppressive regulatory T cells are abundant in the reactive lymphocytes of Hodgkin lymphoma[J]. Blood. 2004, 103: 1755-1762.
25. Samanta M, Iwakiri D, Takada K.Epstein-Barr virus-encoded small RNA induces IL-10 through RIG-I-mediated IRF-3 signaling[J]. Oncogene. 2008 Mar 24 [Epub ahead of print]
26. Irons RD, Le AT.Dithiocarbamates and viral IL-10 collaborate in the immortalization and evasion of immune response in EBV-infected human B Iymphocytes[J]. Chem Biol Interact.2008, 172(1): 81-92.
27. da Silva GN, Bacchi MM, Rainho CA, et al.Epstein-Barr virus infection and single nucleotide polymorphisms in the promoter region of interleukin 10 gene in patients with Hodgkin lymphoma[J]. Arch Pathol Lab Med. 2007, 131 (11): 1691 -6.
28. Marshall NA, Culligan DJ, Tighe J, et al.The relationships between Epstein-Barr virus latent membrane protein 1 and regulatory T cells in Hodgkin's lymphoma[J]. Exp Hematol. 2007,35(4): 596-604.
29. 路慧丽,俞眉,韩伟.趋化因子CXCL9/Mig的研究进展中国生物工程杂志, 2006,26(10):59-63.
30. Yao TC , Kuo ML , See LC , et al . The RANTES promoter polymorphism: a genetic risk factor for near-fatal asthma in Chinese children[J] . J Allergy Clin Immunol,2003,l 11 (6):1285- 1292.
31. Muller K, Bischof S, Sommer F, et al.Differential production of macrophage inflammatory protein 1 gamma (MIP-lgamma), lymphotactin, and MIP-2 by CD4(+) Th subsets polarized in vitro and in vivo[J]. Infect Immun. 2003, 71(11): 6178-83.
32. Reynolds GM, Billingham LJ, Gray LJ, et al. Interleukin 6 expression by Hodgkin/Reed-Sternberg cells is associated with the presence of 'B' symptoms and failure to achieve complete remission in patients with advanced Hodgkin's disease[J]. Br J Haematol. 2002,118(1): 195-201.
33. Kobayashi K, Yokote T, Akioka T, et al. Vascular endothelial growth factor and interleukin 6 production by Hodgkin lymphoma[J]. Gan To Kagaku Ryoho. 2007, 34(8): 1327-30.
34. Smyth DC, Kerr C, Li Y, et al. Oncostatin M induction of eotaxin-1 expression requires the convergence of PI3'K and ERK1/2 MAPK signal transduction pathways[J]. Cell Signal.2008 Feb 11 [Epub ahead of print]
35. Mukherjee A, Rotwein P. Insulin-like growth factor binding protein-5 in osteogenesis:facilitator or inhibitor? Growth Horm IGF Res. 2007, 17(3): 179-85.
36.Strohbach C,Kleinman S,Linkhart T,et al.Potential involvement of the interaction between insulin-like growth factor binding protein(1GFBP)-6 and LIM mineralization protein (LMP)-I in regulating osteoblast differentiation[J].J Cell Biochem.2008 Apr 7[Epub ahead of print]
37.Pazaitou-Panayiotou K,Kelesidis T,Kelesidis I,et al.Growth hormone-binding protein is directly and IGFBP-3 is inversely associated with risk of female breast cancer[J].Eur J Endocrinol.2007,156(2):187-94.
38.Fu P,Thompson JA,Bach LA.Promotion of cancer cell migration:an insulin-like growth factor(IGF)-independent action of IGF-binding protein-6[J].J Biol Chem.2007,282(31):22298-306.
39.Shimaoka T,Seino K,Kume N,et al.Critical role for CXC chemokine ligand 16(SR-PSOX)in Th1 response mediated by NKT cells[J].J Immunol.2007,179(12):8172-9.
40.Kobayashi K,Yokote T,Akioka T,et al.Vascular endothelial growth factor and interleukin 6 production by Hodgkin lymphoma[J].Gan To Kagaku Ryoho.2007,34(8):1327-30.