【作者】 刘繁荣；

【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， 2011， 博士

【摘要】 研究背景经典型霍奇金淋巴瘤(classical Hodgkin lymphoma, cHL)是组织形态非常特殊的一类淋巴瘤,其肿瘤细胞——H/RS细胞约占肿瘤组织的1/100,该细胞Kuppers等通过单细胞分析和IgH基因重排证实它起源于生发中心残疾B细胞,背景的炎症免疫细胞(包括T细胞、B细胞、浆细胞、中性粒细胞、嗜酸性粒细胞和肥大细胞等)及其细胞因子是H/RS细胞赖以生存的基础条件。因此,研究cHL既要注意肿瘤细胞H/RS,还要注重其背景免疫细胞及其细胞因子构成的微环境。H/RS细胞周围微环境的炎症免疫细胞对H/RS细胞的生存是必需的,其炎症免疫细胞以CD4+T淋巴细胞为主,已有证据表明H/RS细胞生存明显依赖其周围细胞释放的生存信号：背景细胞死亡,H/RS细胞消亡。因此,原发肿瘤的H/RS细胞在体外极难培养；HL初发在淋巴结,建立H/RS细胞与背景细胞共同存在的体系,模拟淋巴结内肿瘤状态及生存环境,则是理想的细胞系。但目前均无来自cHL淋巴结的瘤细胞系,且屈指可数的细胞系均来自晚期HL患者的外周血、骨髓或腹水,如L428, L540, L591, DEV, HD-LM2, KM-H2,这些细胞系虽然不能模拟淋巴结内肿瘤状态,但提示H/RS细胞可以离开淋巴结生存；当H/RS细胞转移到其它器官中,背景细胞依然类似淋巴结内病变；H/RS细胞在异种移植的免疫正常小鼠中很难生存(免疫排斥所致),H/RS细胞移植到严重免疫缺陷SCID鼠可以存活与增生,形成移植瘤,但该瘤无cHL的免疫背景细胞,所以目前国际上没有真正体现cHL特点的动物模型。与此同时,cHL必需依赖趋化因子募集免疫背景细胞。本课题组前期经生物信息学分析cHL与弥漫性大B细胞淋巴瘤(diffuse large B cell lymphoma, DLBCL)趋化因子的差异,发现cHL高表达的趋化因子有RANTES/CCL5、CCL17、CCL22等,在复杂的因子网络关系中RANTES处于较中心的位置,H/RS细胞依赖趋化因子RANTES对CD4+T细胞等背景细胞产生趋化作用。国外和我们前期的实验表明cHL微环境细胞中最显著的是CD4+T细胞,特别是Treg (CD4+ CD25+ FOXP3+T细胞)细胞的重要作用,它和CD4+T细胞一起能抑制cHL微环境中的细胞毒性T淋巴细胞,从而有利于cHL的形成。国外研究表明,cHL的发生、发展和NF-κB活性异常增高有关,我们前期的研究结果表明H/RS细胞为CD99基因低表达和NF-κB信号持续活化。本课题组前期构建的mCD99L2-A20细胞亚系,低表达鼠CD99基因,经鉴定其的确具有H/RS细胞的表型,然而mCD99L2-A20细胞RANTES的表达和NF-κB活性如何?这些方面是否与H/RS相类似?要想完整准确地研究cHL,最可行的就是建立类似人cHL病理特征的活体实验动物模型,满足模拟cHL背景环境的细胞多样性和因子复杂性,从而在此背景微环境基础上才能进行cHL的相关研究,课题组前期将mCD99L2-A20细胞尾静脉注射BALB/c小鼠未能成瘤,所以构建HL动物模型陷入了困境。本研究考虑到cHL为免疫系统恶性肿瘤,其发生与机体免疫缺陷有着密切的关系,糖尿病能降低机体免疫力,有研究证实1型糖尿病并发淋巴瘤的风险较正常人高(男性高8%,女性高12%)。若将来自BALB/c鼠的mCD99L2-A20细胞接种同类糖尿病鼠,既能避免种群排斥,又能避免健康BALB/c鼠的免疫排斥,还能提供处于免疫抑制状态的免疫炎症细胞来源,可以用来成瘤并有助于反应背景细胞的募集。为此,本研究采用荧光定量、共聚集显微镜、免疫细胞与免疫组织化学、ELISA、共培养实验、Western blotting、流式细胞术、体内成瘤的方法,首先观测了cHL细胞及瘤组织的RANTES的表达,探究RANTES在cHL形成中的作用；同时检测RANTES在类人H/RS细胞mCD99L2-A20细胞的表达,进行同源BALB/c糖尿病鼠接种形成移植瘤,以期获得鼠源性的早期类人cHL模型。目的1.观测cHL和DLBCL细胞及瘤组织RANTES的表达的情况,比较分析RANTES在cHL形成中的作用。2.检测HL细胞株L428细胞上调CD99基因前后(L428与L428-CD99)、鼠DLBCL细胞株A20细胞下调CD99基因前后(A20与mCD99L2-A20) RANTES的表达,探究RANTES表达与CD99基因及NF-κB信号通路的关系。3.构建链脲佐菌素(Streptozotocin,STZ)诱导BALB/c小鼠1型糖尿病模型,观测外周血RANTES分泌水平。4.比较mCD99L2-A20类人H/RS细胞株在1型糖尿病BALB/c小鼠和正常BALB/c小鼠中成瘤情况,观测外周血中RANTES分泌水平。方法1.HL和DLBCL中RANTES表达的检测HL细胞株L428与DLBCL细胞株OCI-Ly1、OCI-Ly8、OCI-Ly10细胞进行细胞培养,观察细胞的形态,提取RNA,荧光定量检测RANTES mRNA的表达,共聚集显微镜检测细胞内RANTES蛋白的表达,免疫细胞化学检测细胞、免疫组织化学检测HL与DLBCL组织的RANTES蛋白表达情况,IPP6.0软件分析阳性细胞光密值数据,酶联免疫吸附实验(ELISA)检测细胞分泌的RANTES蛋白,共培养实验观测L428分泌的RANTES对CD4+的趋化作用。2. RANTES与CD99、NF-κB信号通路的关系L428与L428-CD99、A20与mCD99L2-A20细胞培养,观察细胞的形态：提取RNA,荧光定量检测CD99mRNA、RANTES mRNA的表达；共聚集显微镜检测细胞内CD99、RANTES蛋白的表达；ELISA检测细胞RANTES蛋白的分泌,Western blotting检测CD99和p-iκBa蛋白的表达。3. BALB/c小鼠1型糖尿病模型的建立及外周血RANTES蛋白的检测将60只雄性BALB/c小鼠随机分为A、B、C、D四组,每组15只,A组采用一次性腹腔注射150mg/kg STZ, B组采用一次性腹腔注射与A组等量无菌柠檬酸缓冲液,C组采用200 mg/kg STZ分5次腹腔注射,D组采用与C组等量无菌柠檬酸缓冲液分5次腹腔注射,监测不同时点空腹血糖变化,A、B、C、D四组均自由饮食。用流式细胞术检测糖尿病鼠与正常鼠外周血CD4+CD25+Treg细胞数,ELISA检测糖尿病鼠与正常鼠外周血RANTES蛋白的分泌情况。4.1型糖尿病BALB/c小鼠类HL模型的构建将类人H/RS细胞(mCD99L2-A20细胞)经尾静脉注射接种1型糖尿病BALB/c小鼠和正常BALB/c小鼠各20只,观察动物成瘤时间、成瘤率、瘤体生长速度；取瘤组织做HE染色观察病理学形态、免疫组化检测RANTES的表达；肿瘤组织进行原代培养,DNA-PCR检测载体的整合；流式细胞仪检测成瘤糖尿病鼠与未成瘤糖尿病鼠外周血CD4+CD25+Treg细胞的比例；ELISA检测成瘤糖尿病鼠与未成瘤糖尿病鼠外周血RANTES蛋白的分泌情况。结果1.HL和DLBCL中RANTES的表达(1)以OCI-Lyl为对照,L428、OCI-Ly8和OCI-Lyl 0细胞的RANTES mRNA表达水平分别是OCI-Lyl的8.154±0.809、0.990±0.062和1.008±0.0565倍,L428细胞中RANTES的mRNA的表达高于OCI-Lyl、OCI-Ly8和OCI-Ly10细胞,两者相比较均有统计学差异(P=0.000); OCI-Lyl与OCI-Ly8 (P=0.959)、OCI-Ly1与OCI-Ly10 (P=0.968)、OCI-Ly8与OCI-Ly10 (P=0.927),两两相比较均无统计学差异。(2)L428细胞中RANTES的表达高于OCI-Ly1、OCI-Ly8或OCI-Ly10细胞,两者相比较均有统计学差异(P=0.000); cHL组织中RANTES的表达高于DLBCL,两者相比较均有统计学差异(P=0.000); cHL组织中RANTES的表达高于L428细胞中的表达,两者相比较均有统计学差异(P=0.000); DLBCL组织中RANTES的表达高于OCI-Ly1、OCI-Ly8或OCI-Ly10细胞中的表达,两者相比较均有统计学差异(P=0.000);OCI-Ly1与OCI-Ly8(P=0.811)、OCI-Lyl与OCI-Ly10 (P=0.279)、OCI-Ly8与OCI-Ly10 (P=0.397),两两相比均无统计学差异。(3)L428细胞中RANTES的分泌高于OCI-Ly1)、OCI-Ly8或OCI-Ly10细胞,两者相比较均有统计学差异(P=0.000); OCI-Ly1与OCI-Ly8(P=0.885)、OCI-Ly1与OCI-Ly10(P=0.979)、OCI-Ly8与OCI-Ly10(P=0.906),两两相比较均无统计学差异。(4)与L428共培养时趋化的CD4+T细胞(340.556±15.852),高于与OCI-Ly1共培养能趋化的CD4+T细胞(79.333±10.782),两者相比有统计学意义(P=0.000)；同时若向L428培养基中加RANTES,可导致趋化的CD4+T细胞增加(571.111±14.049),若向L428培养基中加anti-RANTES抗体,可导致趋化的CD4+T细胞减少(149.222±16.092),两者相比有统计学意义(P=0.000)。2. RANTES与CD99、NF-κB信号通路的关系(1)在传代过程中发现与A20细胞相比较,mCD99L2-A20细胞的大细胞数量明显增多,胞浆较丰富,细胞核明显增大,出现较多双核、多核细胞；与L428细胞相比较,L428-CD99细胞中的大细胞数量明显减少,细胞更具有一致性。(2) RT-PCR及荧光定量PCR检测：mCD99L2-A20细胞的mCD99基因,为A20细胞的0.519±0.004倍,呈低水平表达,两者相比有统计学意义(P=0.000); mCD99L2-A20细胞的mRANTES mRNA,为A20细胞的15.224±0.984倍,两者相比有统计学意义(P=0.000);L428-CD99细胞的hCD99基因是L428细胞的24.503±0.681倍,两者相比有统计学意义(P=0.000); L428-CD99细胞的hu-RANTES mRNA较L428细胞减少80.932±2.062倍,两者相比有统计学意义(P=0.000)。(3)荧光共聚集检测：mCD99L2ˉA20细胞内RANTES蛋白的表达高于A20细胞,L428-CD99细胞内RANTES蛋白的表达低于L428细胞；(4) Western blotting检测：mCD99L2ˉA20细胞mCD99蛋白弱于A20细胞,p-iκBα蛋白强于A20细胞；mCD99L2ˉA20细胞经NF-κB信号通路抑制剂BAY处理对mCD99蛋白无影响,但p-iκBα蛋白表达明显下降；A20细胞经NF-κB信号通路激活剂LPS处理对mCD99蛋白无影响,但p-iκBα蛋白表达明显增强。L428细胞hCD99蛋白弱于L428-CD99细胞；L428细胞经NF-kB信号通路抑制剂BAY处理对hCD99蛋白无影响,但p-iκBα蛋白表达明显下降；L428-CD99细胞经NF-κB信号通路激活剂LPS处理对hCD99蛋白无影响,但p-iκBα蛋白表达明显增强。(5) mCD99L2ˉA20细胞分泌RANTES的量为432.722±10.735,高于A20细胞分泌RANTES的量39.343±1.819,两者相比具有统计学意义(P=0.000,),且mCD99L2ˉA20细胞经NF-kB信号通路抑制剂BAY处理后其分泌RANTES量为116.391±6.241,比处理前明显减少,两者相比具有统计学意义(P=0.000)；A20细胞经NF-kB信号通路激活剂LPS处理后其分泌RANTES量为246.838±7.163,比处理前明显增加,两者相比具有统计学意义(P=0.000)。L428-CD99细胞分泌RANTES的量为44.612±3.228,低于L428细胞分泌RANTES的量839.388±2.605,两者相比具有统计学意义(P=0.000)；且L428细胞经NF-κB信号通路抑制剂BAY处理后其分泌RANTES量为109.107±5.100,比处理前明显减少,两者相比具有统计学意义(P=0.000); L428-CD99细胞经NF-kB信号通路激活剂LPS处理后其分泌RANTES量为443.618±12.514,比处理前明显增加,两者相比具有统计学意义(P=0.000)。3. BALB/c小鼠1型糖尿病模型及其外周血RANTES表达(1)A组糖尿病成模率为86.67%,C组糖尿病成模率为93.33%,血糖均在3w后稳定在较高水平且无明显下降,两组糖尿病模率相比无统计学差异(P=1.000)。(2)糖尿病成模组与对照组外周血CD4+ CD25+ Treg分别为4.059%±0.384%和8.576%±0.536%,两组相比较有统计学意义(P=0.000)(3)糖尿病成模组与对照组外周血RANTES分别为124.551±3.683 pg/ml和87.222±6.707 pg/ml,两组相比较有统计学意义(P=0.000)4.糖尿病鼠类人霍奇金淋巴瘤荷瘤模型及RANTES表达(1) mCD99L2-A20细胞成瘤情况：20只1型糖尿病BALB/c小鼠,成瘤6只(成瘤率30%),成瘤于腋下或腹股沟淋巴结,成瘤时间为34.67±2.16d；20只正常BALB/c小鼠均未成瘤。(2)糖尿病BALB/c小鼠mCD99L2-A20细胞成瘤生长速度,慢于A20细胞接种正常BALB/c鼠成瘤,两者相比有统计学意义(P=0.000)。(3)病理变化：肉眼观切面灰白,质实；镜下大细胞散在分布于被膜下淋巴窦和髓窦,其周围为小淋巴细胞浸润,与cHL早期形态相似；免疫组化检测背景CD3阳性细胞数多于CD20阳性细胞数。(4) DNA-PCR检测糖尿病BALB/c小鼠瘤组织中有mCD99L2-A20干扰载体。(5)外周血CD4+ CD25+Treg细胞,成瘤糖尿病鼠为31.293%±1.573%,高于未成瘤糖尿病鼠4.493%±0.238%,两者相比有统计学意义(P=0.000)(6)外周血RANTES成瘤糖尿病鼠为257.293±5.696,高于未成瘤鼠RANTES 124.342±3.882,两者相比有统计学意义(P=0.000)。结论1.H/RS细胞高分泌RANTES有利于趋化更多的CD4+T细胞。2. mCD99L2- A20类H/RS细胞RANTES为较高表达,其RANTES的分泌受NF-kB信号通路的调控。3.类H/RS细胞糖尿病鼠荷瘤模型初步具有人早期cHL淋巴瘤的一些特征。4.类人霍奇金淋巴瘤荷瘤模型外周血RANTES含量明显增加。创新之处1.本研究从cHL的微环境入手,对比检测了cHL和DLBCL细胞和组织中RANTES表达的差异,论证了高分泌RANTES的cHL细胞对CD4+T细胞的具有更强的趋化作用,利于cHL的形成。2.证实了本课题组构建的mCD99L2-A20细胞亚系具有与H/RS细胞相似的特性,高表达RANTES;巧妙地运用1型糖尿病BALB/c小鼠免疫力受抑制和高表达RANTES的特点,首次探索经鼠尾静脉注射mCD99L2-A20细胞,获得了成瘤。3.构建的糖尿病鼠类人早期cHL模型位于淋巴结内,背景细胞以T淋巴细胞为主,高表达RANTES,为进一步调控RANTES表达构建典型的类人cHL动物模型奠定一定的理论和实验基础。更多还原

【Abstract】 BackgroudClassical Hodgkin lymphoma(cHL) is a very special form tumor, whose tumor cells are H/RS cell that accounted for one percentage of parenchymal cells. Up to ten years ago, Kuppers confirmed that H/RS derived form B lymphocyte with single cell analysis and IgH gene rearrangement. There are lots of immune cells (including T lymphocytes, B lymphocytes, Plasm cells, Neutrophils, Eosinophils and Mast cells, etc.) and cytokines, which is the basic conditions for survival of H/RS cells. It is apparent that we must notice not only H/RS cell but also the microenvironment of immune cells and cytokines on the research of Hodgkin lymphoma.The microenvironment surrounding H/RS cells is necessary for survival of H/RS cells. Recent years tested that the survival of H/RS cells depends on the signal of microenvironment surrounding H/RS cells, that is to say, if the background cells die, H/RS cells must die, so H/RS cell of primary tumor is extremely difficult to culture in vitro. Because of Hodgkin lymphoma onset in the lymph nodes depending on lymph node status and survival of tumor environment, it’ll be an ideal cell line of co-exist of HRS cells and background cells. At present there is a few H/RS cell lines were derived from the peripheral blood, bone marrow or ascites, not came from lymph nodes of HL patient’s, including L428, L540, L591, DEV, HD-LM2 and KM-H2, Although these cells cannot copy the state of HL nodes, H/RS cells can live in other organs except nodes during the late phase of HL. There is few H/RS cells in the peripheral blood of early HL patient’s. There are still lots of inflammatory cells surrounding H/RS cells, which is similar to lesion of HL nodes. H/RS cells cannot exist in normal mice for immune rejection. H/RS cells can exist in severe combined immunodeficiency mice to form tumor without background of nonneoplastic inflammatory cells, there is not HL animal models that have the characteristics of HL at present.At the same time chemokines may not only be involved in the attraction of other cells into the lymphoma microenvironment but also have direct effects on H/RS cells survival and proliferation. Our results suggested that the defferent chemokines exist in cHL and Diffuse Large B cell Lymphoma(DLBCL) by Bioinformatics analysis, including RANTES, CCL17 and CCL22 etc of HL. RANTES is in a relatively central location Complex cytokine network diagram. Studies have shown that H/RS cells attract CD4+ background cells dependenting the RANTES chemokine. Foreign and our previous experimental results tested that the most number of cells in HL microenvironment cells is CD4+T cells. CD4+ CD25+regulatory T cells can hold Cytotoxic T lymphocytes back, which play a very important role in formation of cHL too.Foreign studies have shown that the activity abnormal increasing of NF-κB is related to the occurrence and development of cHL. our previous results show that the H/RS cells with low CD99 gene expression and sustained activation of NF-κB signaling. Pre-constructed by our group mCD99L2-A20 cell line, low expression of mouse CD99 gene, identified its does have a H/RS cell phenotype, but How mCD99L2 A20 cell RANTES expression and activity of NF-κB is? Whether is the cell similar to H/RS?In order to research cHL clearly, the best way is to establish cHL animal model similar to cHL patient’s, which may meet the research about cHL, a variety of background cells and cytokines supporting H/RS cells survival, proliferation and apoptosis suppression. There is no tumor formation by intravenous injection mCD99L2-A20 cells during our previous research. therefore, construction of animal models of HL falled into a plight. In view of HL belongs to the immune system disease, immune defects are closely related. Diabetes can suppress the body’s immune system, research has shown that type 1 diabetes complicated by higher risk of lymphom than normal people. Therefore, If diabetic BALB/c mice is inoculated by lymphoma cell line of similar mice. Not only avoid exclusion of population, but also avoid immune rejection of health BALB/c mice. Also provides inflammatory cells in a state of immune suppression. It benefits to form tumor and useful to raise the background reaction in tumor tissue.In this research, Fluorescent quantitative PCR(QPCR), confocal microscopy, Immunocytochemistry(ICC), immunohistochemistry(IHC), enzyme linked immunosorbent assay(ELISA), Co-culture experiments, Western blotting, Flow cytometry and formation tumor in vivo were used. At first, the expression of RANTES in HL and DLBCL, analysed the role of RANTES to HL formation. Detect the expression of RANTES in mCD99L2-A20 cell, inoculated by intravenous injection of diabetic BALB/c mice to tumor, similar to cHL mouse model, and analysed the role of RANTES in this model.Objective1. Explore the expression of RANTES in Hodgkin lymphoma and diffuse large B cell lymphoma cells and tissue, and analyse the role of RANTES to form Hodgkin lymphoma.2. Detect the expression of RANTES in L428, L428-CD99, A20 and mCD99L2-A20 cells. Analyse the relation RANTES expression and CD99 gene.3. Construct type 1 diabetic BALB/c mice and detect the expression of RANTES chemokine in these mice peripheral blood.4. Compare the tumor of mCD99L2-A20 cells in BALB/c mice and type 1 diabetes BALB/c mice, and analyse the expression of RANTES chemokine in these mice peripheral blood.Method1. Detecting the expression of RANTES in Hodgkin lymphoma and diffuse large B cell lymphomaHL cell line L428 and diffuse large B cell lymphoma cell lines Lyl, Ly8, Ly10 cells were cultured. Observing these cell morphology, extracting RNA, analysing RANTES mRNA by RT-PCR and real time RT-PCR, detected RANTES protein by confocal Microscope. Detecting the RANTES expression in cells and tissue of HL and DLBCL by Immunocytochemistry and Immunohistochemistry, analysing the optical density value data of RANTES protein. detecting the secreted RANTES protein by enzyme linked immunosorbent(ELISA), detecting the chemotaxis of CD4+ cell by co-culture experiments with L428 cell.2. Analysing the relation of RANTES and CD99 and NF-κB signal pathwayL428 and L428-CD99, A20 and mCD99L2-A20 lymphoma cell lines were cultured. Observing these cell morphology, RNA extraction, analysing RANTES mRNA by RT-PCR and real time RT-PCR, detecting RANTES protein by confocal Microscope. detecting the RANTES expression in cells and tissue of HL and DLBCL by Immunocytochemistry and Immunohistochemistry, analysing the optical density value data of RANTES protein. detecting the secreted RANTES protein by enzyme linked immunosorbent(ELISA), detecting the expression of CD99 and p-iκBa protein by Western blotting.3. Constructing BALB/c mouse model of type 1 diabetes and detecting RANTES60 male BALB/c mice were randomly divided into A, B, C, D four groups, n= 15, A group were administered intraperitoneally with 150 mg/kg STZ, B group was with the A group by intraperitoneal injection of equal sterile citrate buffer, C group were 200 mg/kg STZ intraperitoneally 5 times, D group and the C group with the same amount of sterile citrate buffer 5 times by intraperitoneal injection, monitoring changes in fasting blood glucose at different time points, A, B, C, D four groups were free diet. CD4+CD25+Treg cells were detected by flow cytometry in peripheral blood of diabetic mice and normal mice. RANTES protein secretion of peripheral blood of diabetic mice and normal mice was detected by ELISA, respectively.4.Constructing type 1 diabetes in BALB/c mice with cHL-like modelmCD99L2-A20 cells that are silimar to H/RS cells were inoculated intravenously with type 1 diabetes BALB/c mice and normal BALB/c mice were each 20 to observe the animals, the time of tumor, tumor rate, subcutaneous tumor growth, take HE staining tumor tissues pathology, the expression of RANTES is detected by immunohistochemistry, tumor tissues were cultured, DNA-PCR detection of vector integration, CD4+ CD25+ Treg cells were detected by flow cytometry in peripheral blood of diabetic mice and normal mice. RANTES protein secretion of peripheral blood of diabetic mice with tumor or no tumor was detected by ELISA. Result1. the expression of RANTES in Hodgkin lymphoma and diffuse large B cell lymphoma(1) OCI-Lyl as a control, the expression of RANTES mRNA in L428, OCI-Ly8 and OCI-Ly10 cells are 8.154±0.809,0.990±0.062 and 1.008±0.0565 folds.The mRNA expression of RANTES in L428 cell is higher than that of OCI-Lyl or OCI-Ly8 or OCI-Ly10 cell, the two is statistically significant (P=0.000), respectively. OCI-Lyl and OCI-Ly8 (P=0.959), OCI-Lyl and OCI-Ly10 (P=0.968), OCI-Ly8 and OCI-Ly10 (P=0.927), compared with no statistical differences.(2) The RANTES protein expression of L428 cells is higher than that of OCI-Lyl, OCI-Ly8 or OCI-Ly10 cells, the expression RANTES protein of cHL tissue is higher than that of DLBCL tissue, the expression RANTES protein of cHL tissue expression is higher than that of L428 cells,the expression RANTES protein of DLBCL tissue is higher than that of OCI-Lyl, OCI-Ly8 or OCI-Ly10 Cells, all have a statistical comparison of the two School differences (P=0.000). OCI-Lyl and. OCI-Ly8 (P=0.811), OCI-Lyl and OCI-Ly10 (P=0.279), OCI-Ly8 and OCI-Ly10 (P=0.397), have no significant difference.(3) The RANTES secretion of L428 cells than that of OCI-Ly1, OCI-Ly8 or OCI-Ly10 cells, comparison of the two were statistically significant (P=0.000). OCI-Lyl and OCI-Ly8 (P= 0.885), OCI-Lyl and OCI-Ly10 (P=0.979), OCI-Ly8 and OCI-Ly10 (P=0.906), compared with no significant difference.(4) when co-cultured with L428 chemotaxis of CD4+T cells (340.556±15.852), higher than the co-culture with OCI-Ly1 can chemotaxis of CD4+T cells (79.333±10.782), comparing the two was statistically significant (P=0.000); the same time if the medium to the L428 plus CCL5, can lead to chemotaxis of CD4+T cells increased (571.111±14.049), if added to the medium anti-CCL5 antibody, can lead to chemotaxis of CD4+T cells Reduction (149.222±16.092), both statistically significant compared (P=0.000).2. the relation of RANTES and CD99 and NF-κB signaling pathway(1) during passage mCD99L2-A20 cells found in the cytoplasm of large cells rich nuclei significantly increased, there are more dual-core, multi-core cell; L428-CD99 cells in the large cells cells cells significantly reduced cell More consistency.(2) RT-PCR and quantitative PCR test:the mCD99 mRNA of mCD99L2-A20 cells was 0.519±0.004 times that of A20 cells, the mRANTES mRNA of mCD99L2-A20 cells was 15.224±0.984 folds that of A20 cells, the hCD99 mRNA of L428-CD99 cells was 24.503±0.681 times that of L428 cells, the two-phase Statistically significant compared (P=0.000), the hu-RANTES mRNA of L428-CD99 cells decreased 80.932±2.062 folds than that of L428 cells, the two compared statistically significant (P=0.000).(3) fluorescence detection:the RANTES protein expression of mCD99L2 A20 cells than that of A20 cells, the RANTES protein expression of L428 cells than that of L428-CD99 cells.(4) Western blotting detection:mCD99L2-A20 cells in mCD99 protein was weaker than A20 cells, p-iκBa protein stronger than A20 cells, mCD99L2-A20 cells by NF-kB signaling pathway inhibitors BAY had no effect on mCD99 protein, but p-iκBa protein expression was significantly decreased, A20 cells by NF-kB signaling pathway activator, LPS had no effect on mCD99 protein, but the p-iκBa protein expression was significantly increased. L428 cells in hCD99 protein was weaker than L428-CD99 cells, L428 cells by NF-kB signaling pathway inhibitors BAY had no effect on hCD99 protein, but the p-iκBa expression decreased, L428-CD99 cells by NF-kB signaling pathway activator LPS treatment had no effect on the hCD99 protein, but the p-iκBa protein was enhanced.(5) the amount of RANTES secretion in mCD99L2-A20 cells was 432.722±10.735, higher than the amount of RANTES secretion in A20 cells 39.343±1.819, both are significant differences (P=0.000).and mCD99L2-A20 cells were NF-kB signaling pathway inhibitor BAY treatment capacity of the secretion of RANTES 116.391±6.241, significantly reduced compared with before treatment, both statistically significant compared (P=0.000), A20 cells by NF-kB signaling pathway activator, LPS RANTES secretion after the amount of 246.838±7.163, significantly increased compared with before treatment, compared to statistical significance between the two (P=0.000). L428-CD99 cells to produce RANTES in the amount of 44.612±3.228, lower than the amount of L428 cells to secrete RANTES 839.388±2.605, both statistically significant compared (P=0.000), and L428 cells were treated with inhibitors of NF-kB signaling pathway after the secretion of RANTES BAY amount 109.107±5.100, significantly reduced compared with before treatment, both statistically significant compared (P=0.000), L428-CD99 cells by NF-kB signaling pathway activator secreted RANTES after LPS volume of 443.618±12.514, significantly increased compared with before treatment, both statistically significant compared (P=0.000).3. Construct BALB/c mouse model of type 1 diabetes(1)It was found that 86.67% and 93.33% male mice were developed diabetes mellitus in group A and group C, respectively. they have no significant difference (P=0.559).(2) CD4+ CD25+ Treg were 4.059%±0.384% in peripheral blood of diabetic mice and 8.576%±0.536% in peripheral blood of normal mice, compared the two groups was statistically significant (P=0.000).(3)RANTES was 124.551±3.683 pg/ml in peripheral blood of diabetic mice and 87.222±6.707 pg/ml in peripheral blood of normal mice, compared the two groups was statistically significant (P=0.000).4.Construction of type 1 diabetes in BALB/c mice with MCD99L2-A20cells into the tumor model(1) mCD99L2-A20 cells into the tumor cases:there are 6 of 20 type 1 diabetic BALB/c mice (30%) formation tumor, tumor in the armpit or groin, the tumor formation time of 34.67±2.16 days. after three months by tail vein injecting 20 normal BALB/c mice, killing mice, with no tumor formation.(2) mCD99L2-A20 cells into tumors of diabetic BALB/c mice grew more slowly than A20 cells tumor of normal BALB/c mouse (preliminary data by our group), there was significant difference between the two groups (P=0.000).(3) pathology:HE section showed that mCD99L2-A20 cells scattered in the lymphocytesbackground, the number of CD3 positive cells in the background was more than the number of CD20 positive cells by immunohistochemical detection.(4) tumor tissue of diabetic BALB/c mice:DNA-PCR detection of mCD99L2-A20 interference vector integrated into the cell genome, RT-PCR gene mRNA levels showed mCD99L2 lower than A20 cells.(5) CD4+CD25+ Treg in peripheral blood of diabetic tumor mice was 31.293%±1.573%, higher than the no tumor diabeticmice 4.493%±0.238%, there was significant between the two groups (P=0.000).(6) RANTES in peripheral blood of diabetic tumor mice was 257.293±5.696, higher than the no tumor diabeticmice 124.342±3.882, there was significant between the two groups (P=0.000).Conclusion1. L428 cells with high secretion of RANTES is conducive to more CD4+T cells. 2. Gene expression and secretion of RANTES of mCD99L2 A20 cell are controlled by NF-kB signaling pathway.3. mCD99L2-A20 cell transplanted tumor model is partly like the characteristics of early cHL.4. RANTES levels are increased in peripheral blood of cHL-like model.New point1. This study compared Hodgkin’s lymphoma with diffuse large B cell lymphoma, detected RANTES expression of cell lines and tissues differences in the two lymphoma, analysed the high secretion of RANTES can chemotaxis more CD4+ T cell, which is conducive to the formation of Hodgkin’s lymphoma.2. This study demonstrated high RANTES expression of mCD99L2-A20cells and the cell was similar to the characteristics of H/RS cells, type 1 diabetes BALB/c mice with immune suppression and high expression of RANTES characteristics was used to HL-like mice model. mCD99L2-A20 cells were injected into by the tail vein inoculation of diabetic mice at first time.3. The cHL-like mice model is the lymph nodes tumor, which is characteristic of T lymphocytes surrounding big cell, and high expression of RANTES. for the further regulating RANTES expression to construct the typical animal models of cHL, which lay a theoretical and experimental basis.更多还原