【作者】 陈小艳；

【导师】 赵彤；

【作者基本信息】 南方医科大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 研究背景霍奇金淋巴瘤（Hodgkin’s lymphoma,HL）是一种好发于儿童和中青年的淋巴造血系统恶性肿瘤,它虽然是恶性淋巴瘤的一部分,但其组织病理学特征与非霍奇金淋巴瘤（non Hodgkin’s lymphoma,NHL）截然不同,其恶性成分--H/RS（Hodgkin/Reed-Sternberg,H/RS）细胞一般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,如此少量的H/RS细胞是如何生存于大量的背景细胞之中的?它与背景细胞究竟存在什么关系?H/RS细胞是如何发生的?这些问题170多年来一直困扰着医学界。要探究这些悬而未决的问题,当务之急是建立类似人HL病理特征的淋巴瘤动物模型,观察H/RS细胞与背景细胞之间的相互作用,以揭示H/RS细胞生存、增殖、免疫逃避、免疫调节的机制,这是本课题组多年来在国家自然科学基金资助下一直致力研究的核心问题。最近十几年来有关H/RS细胞的研究取得了很大的进展,研究结果表明H/RS细胞起源于生发中心凋亡前B细胞（残疾B淋巴细胞）。κ基因结合核因子（NF-κB）是调节B细胞分化和存活的重要转录因子,正常状态下NF-κB仅表现为对不同刺激产生一过性活化,但NF-κB在H/RS细胞中则表现为持续活化。NF-κB的持续活化是H/RS细胞显著的分子特征,它能抑制H/RS细胞凋亡,促进其增殖,故NF-κB被认为是H/RS细胞的存活因子。NF-κB属于一个高度保守的转录因子家族,作为重要的转录调控因子,NF-κB调节很多基因表达,这些基因与细胞增殖、分化、免疫反应、凋亡、转化有着密切关系。现已明确HL的发病机制与EB病毒（Epstein-Barr virus,EBV）感染有关,其潜伏膜蛋白1（latent membrane protein 1,LMP1）通过模拟激活的CD40受体信号而诱导NF-κB的持续活化。LMP1通过活化NF-κB信号传导通路,下调淋巴细胞CD99的表达,可诱导出具有HL病理学特征的H/RS细胞的产生。研究证实,将LMP1基因转染EBV阴性的BJAB细胞和IM9细胞,使CD99蛋白表达减少或缺失的B淋巴细胞显示出了与H/RS细胞类似的表型。本研究组前期将LMP1基因导入小鼠B淋巴瘤细胞株A20细胞后,部分细胞出现了H/RS样细胞的形态特征及免疫表型,初步建立了鼠类人H/RS细胞模型;进一步再利用RNAi技术将A20细胞中的mCD99L2（mouse CD99 antigen-like2）基因沉默,经过在体外反复传代、克隆筛选和鉴定,建立了低表达mCD99L2基因的LV-mCD99L2-A20克隆株,发现其中也出现了类人HL病理学特征的H/RS样细胞。动物体内成瘤实验结果表明,这种低表达mCD99L2基因的LV-mCD99L2-A20克隆株在生物学特性方面与H/RS细胞也具有一定程度的相似性。然而令人遗憾的是,本研究组前期采用传统的脂质体转染法将LMP1基因转染小鼠B淋巴瘤细胞株A20,由于该细胞属于悬浮细胞,转染效率很低,而且所诱导出的H/RS样细胞因在体外培养困难,故未能成功建立长期稳定表达LMP1基因的鼠类人H/RS细胞模型,动物体内成瘤实验也未能成瘤,因而限制了本课题组对HL发病机制研究的开展。目前需要亟待解决的问题是,如何建立长期稳定表达LMP1基因和/或低表达mCD99L2基因的鼠类人H/RS细胞模型?慢病毒载体具有能感染分裂期和非分裂期细胞,使目的基因能在靶细胞内长期稳定表达的优势,而本课题组前期未能成功建立长期稳定表达LMP1基因的鼠类人H/RS细胞模型,本研究拟构建含目的基因LMP1的重组慢病毒表达载体,采用慢病毒转染细胞的技术,将外源基因导入靶细胞A20,在前期课题研究结果的基础之上（LMP1基因导入小鼠B淋巴瘤细胞株A20细胞后,部分细胞出现了H/RS样细胞的形态特征及免疫表型）,建立长期稳定表达LMP1基因的鼠类人H/RS细胞模型。鉴于在EBV阴性的HL细胞株L428表达LMP1后能诱导出更多数量的多核RS细胞的实验研究,以及本课题组前期所建立的低表达mCD99L2基因的LV-mCD99L2-A20克隆株也出现了类人HL病理学特征的H/RS样细胞的形态和免疫表型,本研究拟采用所构建的含目的基因LMP1的慢病毒载体再次转染LV-mCD99L2-A20,观察是否能诱导出更多的多核RS细胞,从而建立长期稳定表达LMP1基因同时低表达mCD99L2基因的鼠类人H/RS细胞模型。另外,本课题组前期将初步构建的表达LMP1基因的小鼠H/RS样细胞皮下接种BALB/c小鼠未能成瘤,再将低表达mCD99L2基因的LV-mCD99L2-A20克隆株皮下接种具有健全免疫功能的BALB/c小鼠,因成瘤率极低,仅为3.6%（2/56）,显著低于A20细胞在其体内的成瘤率75%（21/28）,使我们在构建鼠类人HL病变特征的动物模型方面遇到了另一个挑战。本研究需要解决的第二个关键问题是:如何提高鼠类人HL病理特征的H/RS样细胞在BALB/c鼠体内的成瘤率?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,研究表明几乎所有HL伴有AIDS患者的H/RS细胞均有EBV的感染,提示HL的发生既与EBV感染密切相关,同时也伴有免疫功能缺陷。本研究拟将BALB/c小鼠经X线照射后,在降低其免疫功能的基础上,将所构建的表达LMP1的A20细胞以及表达LMP1同时低表达mCD99L2的LV-mCD99L2-A20克隆株皮下接种放射后的BALB/c小鼠,提高鼠类人H/RS细胞在BALB/c小鼠体内的成瘤率,观测鼠类人H/RS细胞在BALB/c小鼠体内的生物学特性、免疫表型和病理特征,从而建立鼠类人HL病理特征的动物模型。目的1.构建含目的基因LMP1和报告基因copGFP的重组慢病毒表达载体,进行慢病毒包装和滴度测定。2.不同方式转染鼠源性B淋巴瘤细胞株A20,探讨悬浮细胞A20最佳的转染方式,为本研究后续工作奠定基础。3.将LMP1基因导入A20细胞和LV-mCD99L2-A20细胞,诱导其转化为H/RS样细胞,构建长期稳定表达LMP1基因和/或低表达mCD99L2基因的鼠类人H/RS细胞模型--LMP1⁺A20和LMP1⁺/mCD99L2^-A20细胞。4.构建LMP1⁺A20和LMP1⁺/mCD99L2^-A20克隆株荷瘤裸鼠模型和BALB/c小鼠模型。方法1.含目的基因LMP1的重组慢病毒表达载体的构建与鉴定以及慢病毒包装和滴度测定根据慢病毒表达载体pCDF1-copGFP的多克隆酶切位点,设计特异性PCR引物,上下游分别引入BglⅡ和EcoRⅠ,以LMP1阳性质粒为模板,扩增目的基因LMP1全长,将之连接到慢病毒表达载体pCDF1-copGFP的多克隆酶切位点,转化大肠杆菌,挑选阳性克隆,经PCR扩增、质粒小量抽提、限制性酶切鉴定和测序鉴定重组载体pCDF1-LMP1-copGFP。在脂质体介导下将慢病毒包装系统的包装结构质粒pFIV、包膜质粒pVSVG和重组质粒pCDF1-LMP1-copGFP导入病毒包装细胞293FT,收集病毒上清,再用病毒上清转染293FT细胞,荧光显微镜观察293FT细胞绿色荧光的分布,高倍镜下对发荧光的细胞进行计数,根据SBI公司使用手册提供的公式计算病毒上清液的滴度（Tu/ml）:滴度=（稀释因子）×（绿色荧光细胞数/计数的细胞总数）×（被转染的细胞数）/0.5。2.不同方式转染鼠源性B淋巴瘤细胞株A20的探讨分别用传统的脂质体转染技术、新兴发展的Nucleofector^TM核酸转染技术和慢病毒载体介导的转染技术,将重组慢病毒表达载体质粒pCDF1-LMP1-copGFP导入鼠源性B淋巴瘤细胞株A20,荧光显微镜观察A20细胞绿色荧光的分布,高倍镜下对发荧光的细胞进行计数,根据以下公式计算转染效率:转染效率=（绿色荧光细胞数/计数的细胞总数）×100%分析比较这三种技术转染悬浮细胞A20的转染效率,探讨A20细胞最佳的转染方式。3.不同滴度慢病毒转染不同类型细胞转染效率的比较将大容积包装的慢病毒上清液经低温超速离心浓缩,再分别用慢病毒上清液转染293FT、正常鼻咽上皮细胞和A20细胞,以及用浓缩后的慢病毒（10⁷Tu/ml）分别转染293FT、L428和A20细胞,转染5d后,荧光显微镜下观察细胞内绿色荧光的分布,比较分析不同滴度慢病毒转染不同类型细胞的转染效率。4.长期稳定表达LMP1基因和/或低表达mCD99L2基因的鼠类人H/RS细胞模型的构建与鉴定采用经大容量包装和低温超速离心浓缩后的含目的基因LMP1的慢病毒转染A20细胞和LV-mCD99L2-A20细胞,有限稀释法筛选LMP1⁺A20单克隆和LMP1⁺/mCD99L2^-A20单克隆细胞。光学显微镜和透射电镜下观察LMP1⁺A20单克隆和LMP1⁺/mCD99L2^-A20单克隆细胞的形态特征;光镜下测试网格计数法动态监测大细胞（直径≥25μm）。RT-PCR和Western blot分别检测细胞内LMP1mRNA和LMP1蛋白的表达;MTT法检测细胞体外增殖能力;Transwell检测细胞的微侵袭能力;流式细胞仪检测细胞周期及细胞的免疫表型（CD19与CD30）。PCR检测LMP1⁺/mCD99L2^-A20细胞内shRNA干扰载体的整合情况;RT-PCR及荧光定量PCR检测LMP1⁺/mCD99L2^-A20细胞内mCD99L2表达水平。5.LMP1⁺A20和LMP1⁺/mCD99L2^-A20克隆株荷瘤裸鼠和BALB/c小鼠模型构建分别将LMP1⁺A20和LMP1⁺/mCD99L2^-A20克隆株皮下接种裸鼠成瘤,观察动物成瘤时间、成瘤率、皮下肿瘤生长速度;取裸鼠瘤块行石蜡包埋、制备病理组织切片、HE染色观察瘤细胞形态;免疫组织化学染色检测瘤组织内LMP1蛋白的表达;取瘤组织做原代培养,RT-PCR检测原代细胞内LMP1mRNA的表达;RT-PCR检测mCD99L2的表达水平;流式细胞仪检测原代瘤细胞CD19和CD30的表达。再分别将LMP1⁺A20和LMP1⁺/mCD99L2^-A20克隆株皮下接种娇档暮途璛线照射（2Gy）后的同源BALB/c小鼠,做上述检测和鉴定;流式细胞仪检测正常BALB/c小鼠、接种瘤细胞成瘤和未成瘤小鼠外周血中淋巴细胞亚群的比例。6.统计学处理采用统计软件SPSS 13.0对实验结果进行统计学处理和分析。计量资料以均数±标准差（x^-±s）表示。MTT法检测体外细胞增殖能力采用析因设计方差分析比较各组间差异。A20与各组细胞中H/RS样细胞数目的比较采用两因素方差分析（two-wayANOVA）比较组间差异。流式细胞仪检测细胞周期、肿瘤细胞CD抗原表达、外周血淋巴细胞亚群比例数据采用单因素方差分析（One-wayANOVA）比较组间差异,LSD多重比较各组间差异。Transwell法检测细胞体外微侵袭能力和两组之间成瘤时间的比较采用两独立样本t检验进行比较,两组之间成瘤率的比较采用X²检验。在体肿瘤生长曲线的比较采用重复测量方差分析方法处理,若球形检验P＜0.05,拒绝球形检验,用Greenhouse-Geisser进行校正,采用校正后的F值和P值。结果1.含目的基因LMP1的重组慢病毒表达载体的构建与鉴定以及慢病毒包装和滴度测定（1）PCR扩增目的基因LMP1所设计引物位于LMP1全外显子的上下游,包含起始密码子和终止密码子,扩增片段大小为1.2 kb。PCR产物的琼脂糖凝胶电泳见一清晰的特异性扩增条带,其大小与理论预期值相符。（2）LMP1基因重组慢病毒表达载体pCDF1-LMP1-copGFP的构建和鉴定将构建的LMP1基因重组慢病毒表达载体pCDF1-LMP1-copGFP的菌液经PCR扩增,琼脂糖凝胶电泳可见1.2 kb的目的基因LMP1片段,小量抽提重组质粒,经EcoRⅠ和BglⅡ双酶切后,获得与目的基因LMP1 1.2kb一致的清晰条带,而空载体质粒上只有6671 bp的单一片段。（3）DNA测序结果取重组质粒进行DNA测序,结果表明我们所获得的LMP1片段与GenBank所公布的相应序列一致,无碱基缺失及错误。（4）慢病毒包装及滴度测定结果用脂质体将已构建的慢病毒载体系统中3种质粒成分导入病毒包装细胞293FT中,荧光显微镜下见大量绿色荧光,证实已有大量质粒转入293FT细胞,绿色荧光位于细胞的胞膜及胞浆,这与LMP1的表达位置一致。取病毒上清液转染293FT细胞,荧光显微镜下观察细胞内绿色荧光的分布,测定病毒上清液的滴度为1.6×10⁵Tu/ml。2.不同方式转染鼠源性B淋巴瘤细胞株A20（1）脂质体转染通过阳离子脂质体Lipofectamine^TM 2000介导,将重组质粒pCDF1-LMP1-copGFP导入A20细胞,转染24～48h后,荧光显微镜下观察仅见个别细胞有绿色荧光。（2）Nucleofector^TM核酸转染采用Amaxa核酸转染仪,将特定的转染溶液Nucleofector^TM和重组质粒pCDF1-LMP1-copGFP导入A20细胞,转染4～48h后,荧光显微镜下观察可见较多细胞有绿色荧光,转染效率在10%左右,表明Nucleofector^TM核酸转染技术可明显提高A20细胞的转染效率。但是,转染72h后几乎不见绿色荧光细胞,并且细胞死亡率逐渐提高。（3）慢病毒上清液转染用含目的基因LMP1的慢病毒上清液转染293FT、正常鼻咽上皮和A20细胞,转染5d后,荧光显微镜下观察,293FT和鼻咽上皮细胞可见较多的绿色荧光,转染效率约为80%,而A20细胞则有少许细胞有绿色荧光,转染效率＜1%。3.浓缩后的慢病毒转染不同类型细胞转染效率的比较用经过大容积包装和低温超速离心浓缩后的慢病毒（滴度为10⁷Tu/ml）分别转染293FT、L428和A20细胞,其转染效率分别为100%、90%和80%。4.长期稳定表达LMP1基因和/或低表达mCD99L2基因的鼠类人H/RS细胞模型的构建与鉴定（1）采用经低温超速离心浓缩后的含目的基因LMP1的慢病毒分别转染A20细胞和LV-mCD99L2-A20细胞,有限稀释法筛选出LMP1⁺A20细胞单克隆株和LMP1⁺/mCD99L2^-A20细胞单克隆株。转染25d后,倒置显微镜和普通光学显微镜观察,发现细胞出现明显的形态改变,细胞体积增大,直径≥25μm,胞浆丰富,细胞核及核仁明显增大,出现单核、双核和多核H/RS样细胞。（2）透射电镜观察,转型后的LMP1⁺A20细胞体积增大,胞浆丰富,细胞器增多,主要表现为线粒体和内质网增多。最明显的是细胞核增大,核仁明显增大,出现多核和双核细胞。（3）细胞计数结果经两因素方差分析显示,不同细胞组之间差异具有显著性（F=529.733,P=0.000）,三组细胞经LSD多重比较显示,LMP1⁺A20和LMP1⁺/mCD99L2^-A20细胞中H/RS样细胞与对照组A20细胞相比,差异具有显著性（P=0.000）,LMP1⁺A20和LMP1⁺/mCD99L2^-A20细胞中H/RS样细胞数目较A20细胞中的H/RS样细胞多;而LMP1⁺A20与LMP1⁺/mCD99L2^-A20细胞中H/RS样细胞数无统计学差异。（4）RT-PCR检测LMP1⁺A20细胞和LMP1⁺/mCD99L2^-A20细胞内LMP1mRNA表达阳性,而对照组A20细胞表达阴性。（5）Western blot检测LMP1⁺A20细胞内LMP1蛋白表达阳性,而空载体组pCDF-A20细胞和对照组A20细胞表达阴性。（6）MTT检测发现LMP1⁺A20细胞体外增殖能力较空载体组pCDF-A20细胞和对照组A20细胞减慢,差异有显著性（P＜0.05）。（7）Transwell法检测细胞体外微侵袭能力,发现LMP1⁺A20细胞的运动能力比pCDF-A20细胞明显减慢,差异具有显著性（P=0.000）。（8）流式细胞仪检测细胞周期发现LMP1⁺A20、pCDF-A20与A20细胞G1期和S期比例无显著性差异,LSD多重比较结果显示:LMP1⁺A20细胞G₂期比例高于pCDF-A20组和A20组,差异有显著性（P=0.010）。pCDF-A20组与A20组G₂期差异不显著（P=0.966）。（9）流式细胞仪检测细胞的免疫表型,单因素方差分析显示LMP1⁺A20、pCDF-A20与A20三组细胞CD30阳性细胞比例有显著性差异（F=326.125,P=0.000）,CD19阳性细胞比例无显著性差异（F=4.473,P=0.650）。LSD多重比较结果显示LMP1⁺A20细胞CD30阳性细胞比例（72.600±1.990%）高于对照组A20细胞（41.173±1.620%）和pCDF-A20细胞（41.587±1.543%）,差异有显著性（P=0.000,P=0.000）。（10）抽提LMP1⁺/mCD99L2^-A20细胞DNA进行PCR,能检测出ShRNA干扰载体稳定整合至细胞基因组。（11）RT-PCR及荧光定量PCR检测LMP1⁺/mCD99L2^-A20细胞mCD99L2的表达水平低于A20细胞,mCD99L2基因的干扰效率约为50%。5.LMP1⁺A20和LMP1⁺/mCD99L2^-A20克隆株荷瘤裸鼠模型的构建与鉴定（1）成瘤情况:LMP1⁺A20和LMP1⁺/mCD99L2^-A20克隆株2×10⁷细胞分别皮下接种裸鼠3只（6个位点）,成瘤率100%,成瘤时间分别为9.5±2.9d和10.5±1.4d。（2）病理形态观察:光镜下见瘤细胞弥漫分布,大小不一,细胞核大深染,核圆形、卵圆形或不规则形,病理性核分裂像多见;LMP1⁺A20克隆株和LMP1⁺/mCD99L2^-A20克隆株均可见散在分布的胞浆丰富的大细胞,呈双核、多核或不规则核,核仁大,微嗜酸性。这些大细胞极其类似人HL的H/RS细胞形态。（3）免疫组织化学染色:瘤组织内LMP1蛋白表达阳性,阳性定位于细胞膜。（4）取瘤组织做原代培养,RT-PCR检测原代瘤细胞内LMP1mRNA的表达,琼脂糖凝胶电泳可见1.2kb片段;（5）RT-PCR检测LMP1⁺/mCD99L2^-A20细胞内mCD99L2的表达水平,低于对照组A20细胞。（6）流式细胞仪检测LMP1⁺A20和LMP1⁺/mCD99L2^-A20原代瘤细胞CD19和CD30的比例分别为83.6%、52.5%和87.1%、61.2%。6.LMP1⁺A20和LMP1⁺/mCD99L2^-A20克隆株BALB/c小鼠模型的建立（1）成瘤情况:LMP1⁺A20和LMP1⁺/mCD99L2^-A20克隆株2×10⁷细胞分别皮下接种BALB/c小鼠,两株细胞未照射组（n=4）均未成瘤;照射后24h组（n=4）接种LMP1⁺A20细胞成瘤率为100%,接种LMP1⁺/mCD99L2^-A20细胞成瘤率为75%。结果表明将两组瘤细胞接种未经照射的BALB/c小鼠,均未见肿瘤形成,而X线全身照射（2Gy）BALB/c小鼠后24h接种瘤细胞则能显著提高成瘤率。（2）病理形态观察:光镜下见瘤细胞弥漫分布,大小不一,细胞核大深染,核圆形、卵圆形或不规则形,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,微嗜酸性,其中可见“镜影细胞”,在肿瘤细胞周围可见淋巴细胞呈散在或灶性浸润,另还可见浆细胞和组织细胞等背景细胞。（3）免疫组织化学染色:检测瘤组织内瘤细胞LMP1表达阳性,定位于细胞膜和细胞浆。（4）RT-PCR检测原代瘤细胞内LMP1 mRNA的表达,可见1.2kb片段;（5）RT-PCR检测LMP1⁺/mCD99L2^-A20细胞内mCD99L2的表达水平,低于对照组A20细胞。（6）外周血淋巴细胞亚群:对接种LMP1⁺A20细胞和LMP1⁺/mCD99L2^-A20细胞成瘤和未成瘤的BALB/c小鼠外周血进行流式细胞检测CD3、CD4、CD8、CD19阳性淋巴细胞亚群的比例,并与正常BALB/c小鼠（n=4）进行比较。方差分析显示五组间各指标均存在显著性差异。LSD多重比较显示:接种LMP1⁺A20和LMP1⁺/mCD99L2^-A20细胞成瘤鼠和未成瘤鼠间CD3（P=0.000）、CD4（P=0.000）、CD8（P=0.000）、CDl9（P=0.000）阳性细胞比例有显著性差异;成瘤鼠CD3、CD4、CD8低于未成瘤鼠,CD19高于未成瘤鼠,而接种LMP1⁺A20细胞成瘤鼠和接种LMP1⁺/mCD99L2^-A20细胞成瘤鼠间CD3、CD4、CD8、CD19阳性细胞比例无显著性差异。结论1.高滴度慢病毒表达载体可高效转染小鼠B淋巴瘤细胞A20,较脂质体转染法和Nucleofector^TM核酸转染法有明显的优势。2.将LMP1基因导入A20细胞和LV-mCD99L2-A20细胞,能诱导A20细胞转化为H/RS样细胞,建立了长期稳定表达LMP1基因和/或低表达mCD99L2基因的小鼠H/RS样细胞模型。3.将LMP1⁺A20和LMP1⁺/mCD99L2^-A20克隆株分别皮下接种裸鼠和经X线照射后的BALB/c小鼠,建立了LMP1⁺A20和LMP1⁺/mCD99L2^-A20克隆株荷瘤裸鼠模型和BALB/c小鼠模型,后者出现了淋巴细胞、浆细胞和组织细胞等背景细胞,类似人cHL的病理特征。创新之处1.探索出了一种高效转染悬浮细胞A20的方法--高滴度慢病毒转染法。2.将LMP1基因导入A20细胞和LV-mCD99L2-A20细胞,建立了长期稳定表达LMP1基因和/或低表达mCD99L2基因的小鼠H/RS样细胞模型。3.将LMP1⁺A20和LMP1⁺/mCD99L2^-A20克隆株分别皮下接种裸鼠和经放射后的BALB/c小鼠,建立了LMP1⁺A20和LMP1⁺/mCD99L2^-A20克隆株荷瘤裸鼠模型和BALB/c小鼠模型,后者出现了类似人cHL的病理特征,初步建立了类人HL小鼠模型。4.X线照射BALB/c小鼠（2Gy）降低其免疫功能后,便于建立荷瘤动物模型。更多还原

【Abstract】 BackgroundHodgkin’s lymphoma（HL）is a malignant lymphatic hematopoietic tumor,which mainly occurs in children as well as young and middle-aged people,although it is part of malignant lymphoma,the histopathological feature is different from that of non-Hodgkin’s lymphoma（NHL）and its malignant component--H/RS（Hodgkin/ Reed-Sternberg）cells account for only a very small part of tumor tissue（＜1%）,and the rest predominant components are substantial lymphocytes in background cells, how can such a small amount of H/RS cells survive in the context of many of the background cells?What is the relationship between H/RS cells and background cells? And how does H/RS cells originate?These questions have been plaguing the medical profession for more than 170 years.In order to explore the pendent issues,it is imperative to establish a HL animal model similar to pathological characteristics of human lymphoma,to observe the interaction between H/RS cells and the background cells,and to reveal the H/RS cell survival,proliferation,immune evasion,and immune modulation mechanism.These are the core issues which our research group have been devoted to explore over several years funded by the National Natural Science Foundation. The great progress about H/RS cell has been made in the past decade,the majority of research results showed that the H/RS cells originate from pre-apoptotic germinal center B cells（crippled B lymphocytes）.κgene combination of nuclear factor（NF-κB）is an important transcription factor,which could regulate B cell differentiation and survival.NF-κB only presents a transient activation to different stimulation in the normal condition,but it performs sustained activation in H/RS cells.The sustained activation of NF-κB in H/RS cells is the significant molecular characteristics,it can inhibit apoptosis of H/RS cells,promote their proliferation.As a result,NF-κB was considered as a survival factor of H/RS cells.NF-κB belongs to a highly conserved transcription factor family,NF-κB regulates a lot of gene expression as an important transcriptional regulatory factor,and these genes are closely related to the cell proliferation,differentiation,immune response, apoptosis and transformation.It is now clear that the pathogenesis of HL correlates with Epstein-Barr virus infection,and the latent membrane protein 1（LMP1）could induce NF-κB persistent activation by simulating the activated CD40 receptor signaling.LMP1 reduced the expression of CD99 in lymphocytes through activated NF-κB signal transduction pathway,and could induce the generation of H/RS cells with HL pathological characteristics.It was confirmed that EBV-negative BJAB cells and IM9 cells transfected with the LMP1 gene,showed a similar phenotype with H/ RS cells by the reduction or deletion of CD99 protein expression in B lymphocytes.Our study group had ever transfected mouse B lymphoma cell line A20 with LMP1 gene,some of which exhibited the morphological features and immunohistochemical phenotype of H/RS-like cells,an initial mouse H/RS-like cell model similar to human H/RS cell was set up;and then we made A20 cells mouse CD99 antigen-like 2（mCD99L2）gene silent by RNAi technology,after repeated subculture in vitro,clone screening and identification,we set up a LV-mCD99L2-A20 clone with low expression of mCD99L2 gene,and we also found that some of them exhibited H/RS-like cells.Animal tumorigenicity experiments showed that LV-mCD99L2-A20 cloned strains also possessed a certain similarity with H/RS cells in the aspect of biological characteristics.Regrettably,however,our early study group ever transferred LMP1 gene into mouse B lymphoma cell line A20 using traditional lipofection transfection,because A20 cells belong to suspension cells,its transfection efficiency was very low,and the culture of this induced H/RS-like cells in vitro was difficult,so we could not set up mouse H/RS-like cell model with long-term and stable expression of LMP1 gene, animal tumorigenicity experiments in vivo also failed,thus these results restricted our research about the pathogenesis of HL.Urgently the current problem required to be solved was how to set up mouse H/RS-like cell model with long-term and stable expression of LMP1 gene and/or low expression of mCD99L2 gene.Lentiviral vector has the advantages in the transfection of dividing and non-dividing cells,so that the target gene could be expressed stablly in target cells in long-term,and in the early study we could not set up mouse H/RS-like cell model with long-term and stable expression of LMP1 gene,this study intended to construct a LMP1 gene recombined lentiviral vector,and to transfect A20 cells with lentivirus technology,on the basis of the early research results（mouse B lymphoma cell line A20 cells were transfected with LMP1 gene,some cells exhibited H/RS-like cells）, we intended to set up mouse H/RS-like cell model with long-term and stable expression of LMP1 gene.In view of the research that EBV-negative HL cell line L428 could be induced to form a greater number of multinuclear RS cells after LMP1 expression,our early study results showed that the LV-mCD99L2-A20 clone with low expression of mCD99L2 gene also exhibited H/RS-like cells in morphology and immunophenotype,this study intended to transfect LV-mCD99L2-A20 cells with LMP1 gene recombined lentiviral vector again,and to observe whether we could induce more multinuclear RS cells,thereby to build mouse H/RS-like cell model with long-term and stable expression of LMP1 gene and low expression of mCD99L2 genes.In addition,our study group had ever subcutaneously inoculated BALB/c mice with normal immune function with established and identified LV-mCD99L2-A20 clone strains,because animal tumorigenicity rate was very low,3.6%（2/56）, significantly lower than that of A20 cells,its tumorigenicity rate was 75%（21/28）,so we encountered a great challenge in building mouse HL animal model similar to human HL pathological characteristics.In this study,the second key problem required to be solved was how to improve the tumorigenicity rate of mouse H/RS-like cells in BALB/c mice in vivo?Lymphoma is a malignant immune system tumor,its occurrence is closely correlated to immune status.A study showed that H/RS cells in practically all HL patients with AIDS were EBV positive,suggesting that the occurrence of HL not only has close correlation with EBV infection,but also accompanies by defects in immune function.This study intended to irradiate BALB/c mice by X-ray,and to inoculate irradiated BALB/c mice subcutaneously with H/RS-like cells with the expression of LMP1 and/or low expression of mCD99L2 simultaneously.On the basis of the attenuate immune function,we hoped to improve the tumorigenicity rate of mouse H/ RS-like cells in BALB/c mice,and to observe its biological and histopathological characteristics,and immunophenotype,thereby we hoped to set up HL animal model similar to human HL pathological characteristics. Objective1.To construct LMP1 gene recombinant lentiviral expression vector with reporter gene copGFP,and to process the packaging and titer determination of lentivirus2.To transfect mouse B lymphoma cell line A20 with different ways,to explore the best transfection mode of suspension cell A20,and to lay the foundation for following research.3.To transfect A20 cells and LV-mCD99L2-A20 cells with LMP1 gene recombinant lentiviral expression vector,to induce them to transform into H/RS-like cells,and to build mouse H/RS-like cell model with long-term and stable expression of LMP1 gene and/or low expression of gene mCD99L2--LMP1⁺A20 cells and LMP1⁺mCD99L2^-A20 cells.4.To construct LMP1⁺A20 and LMP1⁺/mCD99L2^-A20 nude mice model and BALB /c mice model similar to human HL pathological characteristics.Method1.The construction and identification of LMP1 gene recombinant lentiviral expression vector,and the packing of recombinant lentivirus and titer determination.Designing specific PCR primers according to the multiclonal restriction enzyme site of lentiviral expression vector pCDF1-copGFP,BglⅡand EcoRⅠwere inserted in the upstream and downstream respectively,LMP1-positive plasmid as a template, the full-length LMP1 was amplified and cloned into the multiclonal restriction enzyme site of lentiviral expression vector pCDFl-copGFP,and then transferred into E.coli.Those positive clones were selected by PCR amplification,and then a small amount of recombinant plasmid was extracted,digested by restriction enzyme and identified by DNA sequencing.Mediated by liposome,lentivirus packaging systems including packaging structure plasmid pFIV,peplos plasmid pVSVG and recombinant plasmid pCDF1-LMP1-copGFP were transfected into virus packaging cells 293FT, and then virus supernatant was collected to transfect 293FT cells,the distribution of green fluorescence in 293FT cells was observed under fluorescence microscopy,those green fluorescent cells were counted under high-power microscopy,the titer（Tu/ml） of lentivirus supernant was calculated according to the following formula provided by SBI use manual:titer=（dilution factor）×（number of green fluorescent cells/total number of counted cells）×（transfected cells number）/0.52.The exploration of different transfection modes of mouse B lymphoma cell A20Mouse B lymphoma cell line A20 was transfected with recombinant plasmid pCDFl-LMP1-copGFP by traditional lipofection,Nucleofector^TMtransfection which was newly developed,and by lentiviral transfection technology separately.The fluorescence distribution in A20 cells was observed under fluorescence microscopy, those green fluorescent cells were counted under high-power microscopy,and the transfection efficiency was calculated according to the following calculation formula: transfection efficiency=（number of green fluorescent cells）/（total number of counted cells）×l00%The transfection efficiency of suspension cell A20 was analyzed and compared among the three transfection technologies,and the best transfection method of A20 cells was explored.3.The comparision of transfection efficiency of different types of cells transfected by different titer of lentivirusBulk packaged lentivirus supernatant was concentrated by low temperature ultracentrifugation,and then 293FT,normal nasopharyngeal epithelial cells and A20 cells were transfected with lentiviral supernatant,293FT,L428 and A20 cells were transfected with concentrated lentivirus respectively,after 5 days transfection,the distribution of intracellular green fluorescent was observed under fluorescence microscopy,and the transfection efficiency of different types of cells transfected by different titer of lentivirus was compared and analyzed.4.The construction and identification of mouse H/RS-like cell model with long-term and stable expression of LMP1 gene and/or low expression of gene mCD99L2 LMP1 recombinant lentivirus packaged with large-capacity and ltracentrifugated with low temperature was transferred into A20 cells and LV-mCD99L2-A20 cells, LMP1⁺A20 and LMP1⁺/mCD99L2^-A20 monoclonal cells were selected with a limited dilution,their morphologic characteristics were observed by optical microscope and transmission electron microscope.The large cell（diameter≥25μm） was monitored by grid counting method dynamicly under light microscope.The expression of LMP1 mRNA and LMP1 protein were detected by RT-PCR and Western blot respectively.Cell proliferative capacity in vitro was analyzed by MTT assay;the minimally invasive capacity was detected by Transwell.Cell cycle and immune phenotype（CD19 and CD30）were detected by flow cytometry.The integration of shRNA interference vector in LMP1⁺/mCD99L2^-A20 cells was detected by PCR;and the expression level of mCD99L2 in LMP1⁺/mCD99L2^-A20 cells was detected by RT-PCR and fluorescence quantitative PCR.5.The construction and identification of LMP1⁺A20 and LMP1⁺/mCD99L2^-A20 nude mice and BALB/c mice model.LMP1⁺A20 and LMP1⁺/mCD99L2^-A20 clone strains were inoculated subcutaneously into nude mice respectively,the animal tumorigenicity time and rate and growth rate were observed.The nude mice tumor was paraffin-embedded and prepared into histologic section,and the tumor cells morphology was observed by HE staining,the expression of LMP1 protein in tumor tissue was detected by immunohistochemical staining,and the expression of LMP1 mRNA in primary culture tumor cells was detected by RT-PCR,the expression level of mCD99L2 in primary tumor cells was detectd by RT-PCR,and the expression of CD19 and CD30 in primary tumor cells was analyzed by flow cytometry.LMP1⁺A20 and LMP1⁺/mCD99L2^-A20 clone strains were then inoculated subcutaneously into healthy and irradiated BALB/c mice（2Gy）respectively,the detection and identification were the same as the above.Lymphocyte subsets ratio of peripheral blood in normal BALB/c mice,tumor-bearing and non-tumor-bearing BALB/c mice was analyzed by flow cytometry.6.Statistical analysisThe experimental results were processed and analyzed by SPSS 13.0 statistical software.Measurement data was represented by mean±standard deviation（x^-±s）. Different groups of cell proliferation capacity in vitro by MTT assay were analyzed by the use of factorial design analysis of variance.The number of H/RS-like cells in each groups was processed by two-factor analysis of variance（two-way ANOVA）. The data of cell cycle,CD antigen expression in tumor cells and the proportion of lymphocyte subsets in peripheral blood by flow cytometry was processed by one-way ANOVA,the difference among each groups was analyzed by LSD multiple comparisons.Cell minimally invasive capacity in vitro by Transwell and the tumorigenicity time between the two groups were processed by two independent samples t test,the tumorigenicity rate between the two groups was compared using the X² test.The tumor growth curves in vivo was compared using repeated measures analysis of variance,if the spherical test P＜0.05,it was corrected with Greenhouse-Geisser,the F value and P value were applied after correction. Result1.The construction and identification of LMP1 gene recombinant lentiviral expression vector,and the packaging and titer determination of lentivirus（1）PCR amplification of target gene LMP1LMP1 primer was located in the upstream and downstream of the three exons respectively,including the start and stop codon,the amplified fragment size was 1.2 kb,and the PCR product was analyzed by agarose gel electrophoresis,a clear specific amplification band was seen,and its size was in accordince with the theoretical expected value.（2）The construction and identification of LMP1 gene recombinant lentiviral vector pCDFl-LMP1-copGFPThe E.coli.liquid of constructed LMP1 gene recombinant lentiviral vector pCDFl-LMP1-copGFP was detected by PCR,there was a 1.2kb fragment of target gene LMP1 by agarose gel eletrophoresis.A small amount of the recombinant plasmid was extracted,and digested by BglⅡand EcoRⅠ,a clear band of 1.2kb consistent with target gene LMP1was observed by agarose gel eletrophoresis,and there was only a single fragment of 6671 bp on null vector pCDF1-copGFP.（3）DNA sequencing resultsDNA sequencing results of LMP1 gene recombinant plasmid showed that the LMP1 fragment corresponded with the sequence published in GenBank,and there was no base deletion and error.（4）The results of lentiviral packaging and titer determinationThree kinds of plasmid in lentiviral vector system were transferred into lentivirus packaging cells 293FT by lipofectamine^TM2000,a great deal of green fluorescence was seen under fluorescence microscope.It was confirmed that there was a lot of plasmid transferred into 293FT cells.Green fluorescence was located in cell membrane and cytoplasm,which was consistent with the position of LMP1 expression. 293FT cells were then transfected with virus supernatant,the distribution of intracellular green fluorescent was observed under fluorescence microscopy,the titer of recombinant lentivirus was 1.6×l0⁵Tu/ml.2.Mouse B lymphoma cell line A20 was transfected with different ways（l）LipofectionThe recombinant plasmid pCDFl-LMP1-copGFP was transferred into A20 cells by the cationic liposome Lipofectamine^TM2000-mediated,24-48 h later,there was little green fluorescence seen only in individual cells under fluorescence microscopy.（2）Nucleofector^TMtransfectionA specific transfection solution Nucleofector and recombinant plasmid pCDFl-LMP1-copGFP were transferred into A20 cells by amaxa device,4-48 h later, there was more green fluorescence in A20 cells under fluorescence microscopy,the transfection efficiency was about 10%,indicating that Nucleofector^TMtransfection technology could significantly improve the transfection efficiency of A20 cells. However,72 hours after transfection,green fluorescent cells gradually decreased with increased cell mortality.（3）Lentiviral supernatant transfectionLMP1 recombinant lentivirus supernatant was transferred into 293FT,normal nasopharyngeal epithelial and A20 cells respectively,5 days after transfection,there were a lot of of green fluorescent cells among 293 FT and nasopharyngeal epithelial cells under fluorescence microscopy,the transfection efficiency of them was about 80%,whereas there was little green fluorescent cells in A20 cells,the transfection efficiency of A20 cells was less than 1%.3.The comparision of transfection efficiency of different types of cells by concentrated lentivirus 293FT,L428 and A20 cells were transfected with ultracentrifugation concentrated lentivirus（titer of 10⁷Tu/ml）,their transfection efficiency was 100%,90%and 80% respectively.4.The construction and identification of mouse H/RS-like cell model with long-term and stable expression of LMP1 gene and/or low expression of gene mCD99L2（1）LMP1 recombinant lentivirus packaged with large-capacity and ultracentrifugated with low temperature was transferred into A20 cells and LV-mCD99L2-A20 cells, LMP1⁺A20 and LMP1⁺/mCD99L2^-A20 monoclonal cells were selected with a limited dilution,25 days after transfection,obvious morphological changes of those monoclone cells were observed under inverted and light microscope.Cells volume increased,their diameter was larger than 25μm,and in those large cells there was much more cytoplasm,nuclei and nucleolus increased markedly,multi-and dual-nuclei cells were found.（2）The volume of transformed LMP1⁺A20 cells increased under transmission electron microscope,there was rich cytoplasm in these cells,mitochondria and endoplasmic reticulum increased.The most obvious changes were increased nuclei, nucleolus also increased markedly,multi-and dual-nuclei cells appeared.（3）Cell count results were analyzed by the two-way ANOVA,there were significant difference among the different cell groups（F=529.733,P=0.000）,LSD multiple comparison showed that,there were significant differences among the three groups of cells（P=0.000）,the number of H/RS-like cells in LMP1⁺ A20 cells and LMP1⁺/ mCD99L2^-A20 cells were more than that in the control group A20 cells,however, there were no statistical differences between the number of H/RS-like cells in LMP1⁺A20 cells and LMP1⁺/mCD99L2^-A20 cells.（4）The expression of LMP1 mRNA in LMP1⁺A20 cells and LMP1⁺/mCD99L2^-A20 cells was positive by RT-PCR,whereas that in the control group A20 cells was negative.（5）The expression of LMP1 protein in LMP1⁺A20 cells was positive by Western blot detection,whereas that in the null vector group pCDF-A20 cells and in the control group A20 cells was negative.（6）The proliferation ability of LMP1⁺A20 cells in vitro was slower than that of the null vector group pCDF-A20 cells and control group A20 cells by MTT assay,and there was a significant difference（P＜0.05）.（7）The minimally invasive capacity of LMP1⁺ A20 cells in vitro was slower than that of pCDF-A20 cells by Transwell assay,and the difference was significant （P = 0.000）.（8）Cell cycle analysis by flow cytometry showed that the proportion of Gl phase and S phase had no significant difference among the three groups,however,the proportion of G2 phase in LMP1⁺ A20 cells was higher than that in pCDF-A20 cells and A20 cells by LSD multiple comparison（P =0.010）,there was no significant difference bewteen pCDF-A20 cells and A20 cells（P=0.966）.（9）The cells immunophenotype was detected by flow cytometry,single factor variance analysis showed that the proportion of CD30 positive cells had significant difference（F=326.125,P = 0.000）,whereas that of CD 19 positive cells had no significant difference（F=4.473,P=0.650）among the three groups,LSD multiple comparison showed that the proportion of CD30 positive cells in LMP1⁺ A20 cells （72.600±1.990%）was higher than that in A20 cells（41.173±1.620%）and pCDF-A20 cells（41.587±1.543%）,there was a significant difference（P=0.000, P=0.000）.（10）Extraction of DNA in LMP1⁺/mCD99L2^-A20 cells was performed,and shRNA interference vector in LMP1⁺/mCD99L2^-A20 cells was detected by PCR,it was confirmed that shRNA interference vector could be integrated into the cell genome stablly.（11）The expression level of mCD99L2 gene in LMP1⁺/mCD99L2^-A20 cells was lower than that in A20 cells by RT-PCR and fluorescence quantitative PCR detection, the interferrance efficiency of mCD99L2 gene was about 50%.5.The construction and identification of LMP1⁺A20 and LMP1⁺/mCD99L2^-A20 clone strain tumor-bearing nude mice model.（1）Tumorigenicity circumstance:2×10⁷ LMP1⁺A20 and LMP1⁺/mCD99L2^-A20 cells were inoculated subcutaneously into 3 nude mice（6 sites）respectively,the tumorigenicity rate of the both groups were 100%,and the tumorigenicity time was 9.5±2.9 days and 10.5±1.4 days respectively.（2）Morphological observation:tumor cells exhibited diffuse distribution under light microscope,the sizes of them were different from each other,and large deeply stained nuclei could be seen,the nuclei was round,oval or irregular in shape,and pathologic karyokinesis was eazily found,large cells rich in cytoplasm could be also found scattered in the LMP1+A20 cells,there were dual-and multi-nucleolus,and the nuclei were irregular,large and eosinophilic.More dual-and multi-nucleus large cells could be found among LMP1⁺/mCD99L2^-A20 cells,these cells were very similar to H/RS cells in human HL.（3）Immunohistochemical staining:the expression of LMP1 protein in tumor tissues were positive,positioned in the cell membrane.（4）Taking tumor tissue to do primary cell culture,the expression of LMP1mRNA in primary tumor cells were detected by RT-PCR,there was a 1.2kb fragment detected in agarose gel electrophoresis.（5）The expression level of mCD99L2 gene in primary LMP1⁺/mCD99L2^-A20 cells was lower than that in A20 cells by RT-PCR.（6）Flow cytometry detection results showed that the expression of CD19 and CD30 in primary LMP1⁺A20 and LMP1⁺/mCD99L2^-A20 cells was 83.6%、52.5%and 87.1%、61.2%respectively.6.The construction and identification of LMP1⁺A20 and LMP1⁺/mCD99L2^-A20 clone strain tumor-bearing BALB/c mice model.（1）Tumorigenicity circumstance:2×10⁷ LMP1⁺A20 and LMP1⁺/mCD99L2^-A20 cells were inoculated subcutaneously into BALB/c mice respectively,in no irradiation group（n=4）,there failed to bear tumor,however,the tumorigenicity rate in irradiation group（n=4）inoculated with LMP1⁺A20 cells was 100%,and that inoculated with LMP1⁺/mCD99L2^-A20 cells was 75%respectively.These results indicated that inoculating BALB/c mice without irradiation was not conducive to bearing tumor,however,the tumorigenicity rate was significantly increased by inoculating BALB/c mice treated by X-ray irradiation（2Gy）24 hours ago.（2）Morphological observation:the morphological characteristics of tumor cells were similar to that in nude mice,besides,“mirror image cells”among tumor cells could be found under light microscope,there were some lymphocytes scattered or focal infiltration around the tumor cells,and the other background cells such as plasma cells and monocytes could also be found in tumor tissue.（3）Immunohistochemical staining:the expression of LMP1 protein in tumor tissues were positive,positioned in the cell membrane and cytoplasm.（4）To take tumor tissue to do primary cell culture,the expression of LMP1mRNA in primary tumor cells were detected by RT-PCR,there was a 1.2kb fragment detected in agarose gel electrophoresis;（5）The expression level of mCD99L2 gene in primary LMP1⁺/mCD99L2^-A20 cells was lower than that in A20 cells by RT-PCR.（6）Lymphocyte subsets in peripheral blood:the proportion of CD3,CD4,CD8, CD19 positive lymphocyte subsets in tumor-bearing and non tumor-bearing BALB/c mice inoculated with LMP1⁺ A20 cells and LMP1⁺/mCD99L2^-A20 cells was detected by flow cytometry,and with normal BALB/c mice（n=4）compared. Variance analysis showed that there was a significant difference among the five groups of each indicatrix.LSD multiple comparison showed that,CD3（P=0.000）, CD4（P=0.000）,CD8（P=0.000）,CD19（P=0.000）positive cells proportion between tumor-bearing mice and non tumor-bearing mice inoculated with LMP1⁺ A20 and LMP1⁺/mCD99L2^-A20 cells had a significant difference.CD3,CD4,CD8 positive cell proportion in tumor-bearing mice was lower than that in non tumor-bearing mice,whereas CD19 positive cell proportion was higher than that in non tumor-bearing mice.However,the proportion of CD3,CD4,CD8,CD19 positive cells between tumor-bearing mice inoculated with LMP1⁺A20 cells and LMP1⁺/ mCD99L2^-A20 cells had no significant difference.Conclusion1.High titer lentivirus expression vector could transfect mouse B cell lymphoma A20 with highly efficiency;it has obvious advantages compared to lipofection and Nucleofector^TMtransfection.2.After LMP1 gene was transferred into A20 and LV-mCD99L2-A20 cells,A20 cells could be induced to transform into H/RS-like cells,a mouse H/RS-like cell model with long-term and stable expression of LMP1 gene and/or low expression of mCD99L2 gene was established.3.LMP1⁺A20 and LMP1⁺/mCD99L2^-A20 clone strains were inoculated subcutaneously in nude mice and irradiated BALB/c mice respectively,a H/RS-like cell in tumor-bearing nude mice model was built,and a tumor-bearing BALB/c mice model was also established,which appeared background cells such as lymphocytes, plasma cells and monocytes in tumor tissues,which was similar to histopathological characteristics of human cHL. Innovation point1.Exploring a best transfection method of suspension cells A20 with highly efficiency--high-titer lentiviral transfection method.2.After LMP1 gene was transferred into A20 and LV-mCD99L2-A20 cells,A20 cells could be induced to transform into H/RS-like cells,a mouse H/RS-like cell model with long-term and stable expression of LMP1 gene and/or low expression of mCD99L2 gene was established.3.LMP1⁺A20 and LMP1⁺/mCD99L2^-A20 clone strains were inoculated subcutaneously in nude mice and irradiated BALB/c mice respectively,H/ RS-like cell in tumor-bearing nude mice model and BALB/c mice model were set up, the BALB/c mice model was similar to histopathological characteristics of human cHL.An initial mouse HL model was set up.4.The immune function of BALB/c mice was impaired by X-ray irradiation（2Gy）, which facilitated to build tumor-bearing animal model.更多还原