摘要
研究背景
恶性淋巴瘤是原发于淋巴系统的一组疾病,来源于B淋巴细胞,T淋巴细胞或自然杀伤细胞的非正常克隆性增殖,近年来随着AIDS、器官移植、肿瘤放化疗免疫抑制的应用,发病率急剧升高。
淋巴瘤主要有霍奇金淋巴瘤(Hodgkin lymphoma,HL)和非霍奇金淋巴瘤两大类,两者有不同的病理和临床表现,其中经典型霍奇金淋巴瘤(classicHodgkin lymphoma,cHL)占HL 95%以上,cHL有着相当独特的病理特征,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,背景细胞以T细胞为主,约占70%。H/RS来源B淋巴细胞,有报道其发生与CD99基因表达缺失有关,本课题组前期将慢病毒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mCD99L2~-A20克隆株时发现其中出现类似人cHL的H/RS样细胞(命名LV-mCD99L2~-A20,简称1-1),经过鉴定发现其的确具有H/RS细胞的表型,将A20细胞和mCD99L2~-A20细胞分别接种BALB/c小鼠,A20组皮下成瘤率为75%,而mCD99L2~-A20的皮下成瘤率仅有5%,成瘤率极低,所以构建类人HL动物模型陷入了困境。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,此外,H/RS细胞的生存需要特殊的免疫微环境,尤其是背景T淋巴细胞,因此接下来的研究通过改变小鼠的免疫状况来诱导其成瘤。本研究采取了小鼠全身放射的方式,以此来抑制其免疫功能,改变其T细胞亚群分布,达到提高成瘤率的目的,进一步建立类人cHL动物模型打下基础。
H/RS细胞的产生同其所诱导的免疫微环境密切相关,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。H/RS细胞周围的背景细胞大部分为T细胞,但肿瘤细胞并没有被T细胞杀伤,相反得到了存活,据此推测T淋巴细胞在维持H/RS细胞存活当中起着至关重要的作用,肿瘤细胞同T细胞之间通过某些细胞因子相互作用得以共生,以此营造了H/RS细胞适宜的生存环境。那么究竟背景T细胞与cHL及H/RS细胞之间的关系如何,目前的研究只揭露出冰山一角,相关的文献报道比较少,研究方法也很局限,所以两者具体的相互作用机制至今尚未明确。本研究首先通过改变T细胞亚群成功诱导课题组前期构建的类人H/RS细胞—mCD99L2~-A20细胞BALB/c鼠成瘤,接下来采取了体外模拟H/RS细胞的微环境的方式利用前期课题组构建的类人H/RS细胞株mCD99L2~-A20细胞同其同源的BALB/c小鼠胸腺细胞以及脾脏细胞共同培养,初步探索H/RS细胞同周围背景T细胞之间的关系。此外,为进一步明确肿瘤细胞同微环境中T细胞的相互作用机制,本研究还借助了生物信息学方法,以期从大量的生物信息中探索H/RS细胞同背景T细胞相互作用的网络关系。
目的
1.通过放射的方式改变BALB/c鼠体内T细胞亚型分布,促进mCD99L2~-A20类人H/RS细胞株成瘤,为进一步构建类人cHL动物模型打下基础。
2.mCD99L2~-A20细胞同BALB/c小鼠胸腺细胞以及脾脏细胞共同培养在体外模拟H/RS细胞与T淋巴细胞共生的微环境,探究T细胞与mCD99L2~-A20细胞之间的关系并观察两者共培养对体内成瘤的影响。
3.借助生物信息学方法利用公共芯片数据资源以及相关软件进行分析,比较H/RS细胞同B淋巴瘤细胞基因芯片表达数据差异,探究同H/RS细胞周围背景T淋巴细胞产生关系密切的细胞因子和趋化因子,进一步阐明cHL微环境形成的机制。
方法
1.改变T细胞亚群诱导mCD99L2~-A20细胞BALB/c鼠成瘤
将低表达mCD99L2基因的mCD99L2~-A20细胞亚系经皮下接种BALB/c小鼠,小鼠在接种前接受2Gy放射,观察动物成瘤时间、成瘤率、皮下肿瘤生长速度;取瘤组织做HE染色观察病理学形态;肿瘤组织进行原代培养,肿瘤组织细胞悬液流式细胞仪检测mCD30抗原表达;DNA-PCR检测载体的整合;流式细胞仪检测小鼠外周血CD3、CD4、CD8的比例。
2.mCD99L2~-A20细胞与T淋巴细胞共同培养的研究
mCD99L2~-A20类人H/RS细胞同BALB/c小鼠胸腺细胞以及脾脏细胞共同培养,倒置显微镜观察共培养后肿瘤细胞同淋巴细胞的形态;流式细胞仪检测共培养细胞CD4、CD8、CD25抗原表达;培基中加入IL-2(interleukin-2)和TNF(tumor necrosis factor)两种细胞因子,检测淋巴细胞转化能力;共培养细胞接种BALB/c小鼠皮下,观察其成瘤情况,取脏器做HE染色切片观察病理学形态;流式细胞仪检测小鼠外周血CD4、CD8、CD25的比例;ELISA检测共培养细胞上清液中IFN-γ(IFN-gamma)和IL-10(interleukin-10)的表达。
3.芯片数据探究H/RS细胞同T细胞关系与mCD99L2~-A20蛋白芯片比较分析
以B淋巴瘤细胞作为对照,利用GeneSifter在线软件和BRB-Array Tools对公共基因芯片数据库GEO中的H/RS细胞以及B淋巴瘤细胞基因芯片表达数据进行差异统计学分析,并对这些差异基因进行KEGG(Kyoto Encyclopedia ofGenes and Genomes)通道分析;生物学过程分析;STRING 8.0在线分析细胞因子相关差异基因表达蛋白网络关系,并同前期课题组完成的mCD99L2~-A20细胞和mCD99L2~-A20成瘤组织蛋白芯片差异比对;PubMed数据库对筛选出的差异基因进行文献挖掘,对差异细胞因子进行初步分析。
4.本课题所用统计学方法
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((?)±s)表示。两组成瘤率的比较采用两个独立样本χ~2检验;两组成瘤时间的比较采用两样本t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;流式细胞仪检测血淋巴细胞亚群比例数据采用单因素方差分析(One-way ANOVA)比较各组差异,两组间比较采用LSD多重比较法;两组胸腺重量的比较采用两样本t检验;IL-2和TNF刺激淋巴细胞转化实验结果数据采用单因素方差分析比较各组差异,两组间比较采用LSD多重比较法;ELISA检测共培养细胞上清液IFN-γ和IL-10在48h,60h和5d三个时间点实验组和对照组的差异,实验结果数据采用析因设计的方差分析。
结果
1.改变T细胞亚群诱导mCD99L2~-A20细胞BALB/c鼠成瘤
(1) mCD99L2~-A20接种后成瘤情况:皮下接种2×10~7细胞于20只放射后BALB/c小鼠(20个位点),成瘤率65%,较未处理组(5%)明显提高,差异具有显著性(P=0.000),平均成瘤时间为6.54±0.88天,较接种A20组成瘤时间缩短,差异具有显著性(P=0.000),生长速度也较A20组快,差异具有显著性(P=0.000)。
(2)病理形态:放射后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,类似人HL的H/RS细胞形态。
(3)放射后BALB/c小鼠瘤组织原代培养,肿瘤组织细胞悬液流式细胞仪检测mCD30比例为60.2%;
(4) DNA-PCR检测mCD99L2干扰载体整合在细胞基因组。
(5)流式细胞仪检测小鼠外周血淋巴细胞亚群:各组间CD3、CD4阳性细胞比例无显著差异。统计分析显示各组间CD3~+细胞比例差异无显著性(P=0.116),各组间CD4~+细胞比例差异无显著性(P=0.104),CD8~+细胞比例存在显著性差异(P=0.000),放射后接种mCD99L2~-A20细胞成瘤BALB/c小鼠与正常BALB/c小鼠以及未成瘤BALB/c小鼠相比外周血CD8~+细胞比例明显降低(P=0.000,P=0.001)。
2.mCD99L2~-A20细胞与T淋巴细胞共同培养的研究
(1)共培养过程中倒置显微镜观察:共培养48h后胸腺细胞+mCD99L2~-A20组可见淋巴细胞环绕着肿瘤细胞呈花环状生长;胸腺细胞+mCD99L2~-A20+IL-2组48h和72h分别观察发现随着共培养时间的延长,瘤细胞死亡数目逐渐增多,而对照组胸腺细胞+A20+IL-2组此种现象不明显。
(2)流式细胞仪检测共培养1w后细胞CD4~+CD25~+Treg细胞和CD8~+细胞比例:胸腺/脾细胞+mCD99L2~-A20组CD4~+CD25~+Treg细胞比例(7.8%/8.5%)要明显高于胸腺/脾细胞+A20组(1.1%/1.6%);胸腺细胞+mCD99L2~-A20+IL-2组CD8~+细胞比例(72.0%)要高于不加IL-2组(68.0%),同时也明显高于胸腺细胞+A20+IL-2组(57.8%)。
(3)淋巴细胞转化实验:胸腺细胞+mCD99L2~-A20+IL-2共培养组淋巴细胞转化能力要强于缺乏IL-2组,差异有显著性(P=0.037),反之,胸腺细胞+mCD99L2~-A20+TNF共培养组淋巴细胞转化能力要弱于缺乏TNF组,差异有显著性(P=0.044)。
(4)动物皮下接种共培养细胞成瘤实验:胸腺细胞+mCD99L2~-A20(实验组)和胸腺细胞+A20(对照组)共培养1w后分别皮下接种BALB/c鼠,每组3只,2周后实验组均未成瘤,对照组3只均成瘤;胸腺细胞+mCD99L2~-A20/A20+IL-2(实验组/对照组)共培养24h后分别接种3只BALB/c鼠,6只均未成瘤,分别取胸腺观察,实验组胸腺的体积小于对照组,重量也小于对照组,差异具有显著性(P=0.017),HE染色切片观察到实验组胸腺细胞稀疏,对照组胸腺细胞较正常,细胞排列密集。
(5)流式细胞仪检测接受过共培养细胞皮下注射的BALB/c鼠血:接种后2周检测鼠血液CD4、CD25和CD8的表达,胸腺细胞+mCD99L2~-A20组CD4~+CD25~+Treg细胞和CD8~+细胞比例(分别为3.3%和8.9%)均高于单独接种mCD99L2~-A20组(分别为3.1%和5.2%)。
(6)共培养上清检测IFN-γ和IL-10:胸腺细胞+mCD99L2~-A20共培养组(实验组)和胸腺细胞+A20共培养组(对照组)上清液分别在共培养24h、60h和5d检测IFN-γ和IL-10,共培养24h和60h对照组表达较高IFN-γ,差异具有显著性(P=0.001,P=0.001),24h和60h时对照组表达较高的IL-10,差异具有显著性(P=0.000,P=0.000),5d时实验组表达较高的IFN-γ和IL-10,差异具有显著性(P=0.047,P=0.020)。以上结果表明随着共培养时间的延长,实验组Th1细胞分泌了较多的IFN-γ,Th2细胞分泌了较多IL-10。
3.芯片数据探究H/RS细胞同T细胞关系与mCD99L2~-A20蛋白芯片比较分析
(1)对基因表达谱数据GSE8388分析得出cHL细胞株L428和B细胞淋巴瘤细胞株Raji之间差异表达基因KEGG通路分析显示细胞因子和细胞因子受体相互作用(Cytokine-cytokine receptor interaction)通道和JAK酪氨酸激酶和信号转导子和转录活化子(JAK-STAT)通道是差异最显著的两条通道;免疫生物过程差异表达基因谱分析显示在差异最显著的15个基因中发现了CCL5、CCL22、CCL17和IL-6四个因子,它们同cHL H/RS细胞募集T细胞关系密切,它们的表达均比B细胞淋巴瘤高;差异基因生物学过程分析显示在T细胞激活以及细胞因子合成过程中IL-6均发挥了显著的作用。
(2)对基因表达谱数据GSE12453分析得出cHL瘤组织细胞和弥漫大B细胞淋巴瘤组织细胞之间的差异显著的基因302个,cHL组织细胞和正常淋巴细胞之间的差异显著的基因,共363个,两组差异表达基因交叉比较得出cHL相对于B细胞淋巴瘤发病过程相关的特有基因共48个,其中上调6个,下调42个,包括一种上调的细胞因子,IL-16。
(3)利用STRING 8.0在线分析平台对GSE12453分析得出的cHL组和B细胞淋巴瘤DLBCL组差异基因中最显著的14个细胞因子相关基因进行蛋白功能关系分析,并同前期课题组所做mCD99L2~-A20细胞和mCD99L2~-A20成瘤组织蛋白芯片差异比对,筛选出了同H/RS细背景T细胞产生关系最密切的六种细胞因子以及趋化因子,包括RANTES、MIG、CCL22、CCL17、IL-6以及CXCL16,其中MIG处于它们相互作用的中心位置。
结论
1.类人H/RS样细胞—mCD99L2~-A20细胞在正常BALB/c鼠成瘤率极低,经放射免疫抑制T细胞后得到很大的提高,提示了机体的免疫功能下降以及T淋巴细胞亚型分布发生了改变,细胞毒性T细胞减少和辅助性T淋巴细胞相对增多,有利于介导H/RS细胞T细胞微环境的形成,促进瘤体的形成。
2.通过体外模拟H/RS细胞生存的微环境得出H/RS样细胞—mCD99L2~-A20细胞可以募集大量的CD4~+CD25~+Treg细胞,以此来介导免疫耐受,同时抑制CD4~+Th1和CD8~+CTL对其杀伤作用,Th1、Th2以及CTL可通过IL-2、TNF、IFN-γ和IL-10等一些细胞因子同肿瘤细胞发生相互作用,维持肿瘤细胞内环境的稳态。
3.借助生物信息学方法利用公共芯片数据资源以及相关软件分析HL产生过程中H/RS同背景T细胞相互作用的关系密切的细胞因子和趋化因子有RANTES、MIG、CCL22、CCL17、CXCL16、IL-6、IL-16等,本课题组构建类人mCD99L2~-A20细胞模型及BALB/c小鼠体内成瘤较类似人cHL。
Background
Malignant lymphoma is a group of diseases of lymphatic system,which comes from the abnormal clonal proliferation of B lymphocyte,T lymphocyte and natural killer cells.In recent years a sharp rise of incidence appeared as the application of organ transplantation and immunosuppressive chemotherapy.
Lymphoma contains two categories,Hodgkin's lymphoma(HL) and non-Hodgkin's lymphoma(NHL).They have different pathology and clinical manifestations.Classic Hodgkin's lymphoma(cHL) accounts for more than 95%of HL.cHL has an unique feature,and its malignant component-H / RS(Hodgkin / Reed-Sternberg) cells only take a small part(＜1%)of the tumor tissue.The rest are mainly a large number of lymphocytes,most of them are T cells,accounting for about 70%.It was reported that the appearance of H / RS cells were related to the loss of CD99 gene expression.Our research work have previously transfected the mouse B lymphoma cell line A20,which expressed mouse CD99 antigen-like 2 gene,used letivirus ShRNA vector and constructed successfully subseries of A20 cell line with low mCD99L2 gene expression,named "LV-mCD99L2~-A20".Among the mCD99L2~-A20 cells,giant cells like human cHL H/RS cells were found.After that we identified mCD99L2-A20 cells and found they had some H / RS cells phenotype.Then the A20 cells and mCD99L2~-A20 cells were respectively inoculated subcutaneously into BALB / c mice.Tumor formation rate is 75%in A20 group, while the tumor formation rate is only 5%in mCD99L2~-A20 group.Therefore, construction of animal models of cHL got into trouble.In view of cHL belongs to the immune system disease,immune defects are closely related.In addition,H / RS cells survival needs special immune microenvironment,especially the T lymphocytes,they are the most important reconstituent in the microenvironment.So it is necessary to change immune status of mice to induce tumors.It is efficient to irradiate mice before inoculation to suppress their immune systems and change the distribution of T cell subsets to enhance the rate of tumor formation.We try to establish a stable animal model of cHL by this way.
H / RS cells are closely related to the microenvironment induced by immunization.It is reported that a variety of cytokines probably support H / RS cells survival,proliferation and apoptosis suppression.Most of the cells are T cells in the background of H / RS cells.However,the tumor cells are not killed by the T cells.On the contrary,H/RS cells survive.Thus we speculate that T lymphocytes play an essential role in maintainning H / RS cells survival.T cells create a suitable living environment for H/RS cells by secreting some cytokines.In the current study the specific mechanism of interaction between H / RS cells and T cells are not clear because of lack of an appropriate way.In my study I try to imitate the microenvironment of H / RS cells in vitro by coculturing H / RS like cell line mCD99L2~-A20 cells and thymocytes / spleen cells which are homologous with BALB / c mouse.We try to explore the relationship between H / RS cells and the surrounding T cells preliminarily.In addition,in order to clarify the mechanism of interaction of the tumor cells and T cells in the microenvironment,this study also adopts the biological information analysis to explore interaction network of H / RS cells and T cells.
Objective
1.Irradiating BALB / c mice to change the distribution of T cell subsets before inoculating mCD99L2~-A20 cells to improve the rate of animal tumors formation. Laying the foundation for constructing animal models of cHL.
2.Imitating the microenvironment of H / RS cells in vitro by coculturing H / RS like cell line mCD99L2~-A20 cells and thymocytes/spleen cells.Exploring the relationship between T cells and mCD99L2~-A20 cells.Observing the influence of mCD99L2~-A20 cells and T cells coculture to tumor formation.
3.Taking use of bioinformatics resources as well as public chip data analysis software to compare H / RS cells with B cell lymphoma gene expression data chip differences. Exploring the related cytokines and chemokine of the H / RS cells raising the surrounding T lymphocytes.Elucidating the mechanism of the formation of microenvironment.
Method
1.Inducing tumor formation of mCD99L2~-A20 cells by changing T cell subsets of BALB / c mice
Inoculating subcutaneously mCD99L2~-A20 cells into BALB / c mice,which were accepted 2Gy radiotherapy before inoculation.Recording animal tumorigenicity time;detecting tumor growth rate;observing morphous by HE staining slices; detecting CD30 expression of the cell suspension of tumor tissues with Fluorescence Activated Cell Sorter(FACS) technology;detecting integrated vector by DNA-PCR; detecting CD3,CD4,CD8 ratio in peripheral blood of mouse by flow cytometry.
2.The study of mCD99L2~-A20 cells and T lymphocytes coculture
Coculturing H / RS like cell-CD99L2~-A20 cells and thymocytes/spleen cells of BALB / c mouse,observing coculture cells morphous under the inverted microscope; detecting CD4,CD8,CD25 antigen expression of coculture cells by flow cytometry; testing lymphocyte transformation capacity by IL-2 and TNF stimulation;inoculating subcutaneously coculture cells into BALB / c mice;recording animal tumorigenicity time;detecting tumor growth rate;observing morphous by HE staining slices; detecting CD4,CD8,CD25 ratio in peripheral blood of mouse by flow cytometry; detecting IFN-γand IL-10 in coculture supernatant by ELISA.
3.Exploring the relationship of H / RS cells and T cells by chip data analysis and comparing with the mCD99L2~-A20 protein chip
Taking use of online software GeneSifier and BRB-Array Tools to analyse differences of H / RS cells and B lymphoma cell gene expression data in GEO public database,analysing KEGG(Kyoto Encyclopedia of Genes and Genomes) of these differential genes;analysing biological processes;analysing gene expression differences associated protein network by STRING 8.0 and comparing with pre-made protein chip;screening PubMed database literature.
Result
1.Inducing tumor formation of mCD99L2~-A20 cells by changing T cell subsets of BALB / c mice
(1) Tumor formation after inoculating subcutaneously mCD99L2~-A20 cells into the BALB / c mice:inoculated with 2×10~7 cells into 20 BALB / c mice(20 sites),the tumor formation rate was 65%in radiation group,more than control group(5%),the average time of tumor formation was 6.54±0.88 days,less than the A20 group.The difference of the experimental group and control group was significant(P = 0.000).
(2) Pathology:observated tumor tissue by light microscopy.Tumor cells of various sizes scattered in the cytoplasm,rich in large cells,dual-core,multi-core or irregular nucleus.They were very similar to H / RS cells of people.
(3) The expression of CD30 in the cells from tumor tissues is 60.2%.
(4) Tumor tissue primary culture of radiated BALB / c mice:mCD99L2 interference integrated vector was detected in the cell genome by DNA-PCR.
(5) Flow cytometry detection of lymphocyte subsets:There was no significant difference of CD3 and CD4 positive cells of each group.Statistical analysis showed that among three groups there was no significant difference(P = 0.116) of the proportion of CD3~+ cells.There was no significant difference(P = 0.104) of proportion of CD4~+ cells.There was a significant difference of CD8~+ cell ratio(P = 0.000).Compared to the normal BALB / c mice,radiated BALB / c mice of tumor formation inoculated of mCD99L2~-A20 cells had a higher proportion of CD8~+ cells in peripheral blood(P = 0.000).Compared to the radiated BALB / c mice failed to tumorigenicity,radiated BALB / c mice of tumorigenicity inoculated of mCD99L2~-A20 cells had a higher proportion of CD8~+ cells in peripheral blood(P = 0.001).
2.The study of mCD99L2~-A20 cells and T lymphocytes coculture
(1) Observation of coculture cells under the inverted microscope:after 48h coculture of thymocytes and mCD99L2~-A20 cells we could see thymocytes were growing flower ring around the tumor cells;thymus cells + mCD99L2~-A20 + IL-2 group at 48h and 72h could be seen that tumor cells gradually increase the number of deaths in the control group.However,thymocytes + A20 + IL-2 group did not appear the same phenomenon.
(2) Flow cytometry detection of CD4~+ CD25~+ and CD8~+ cell ratios of coculture cells after 1 week:CD4~+ CD25~+ cell ratio(7.8%and 8.5%) of thymus / spleen cells + mCD99L2~-A20 group were significantly higher than that of thymus / spleen ceils + A20 group(1.1%and 1.6%);the proportion of CD8 ~+cells(72.0%) of thymus cells + mCD99L2~-A20 + IL-2 group was higher than the group without IL-2(68.0%),but significantly higher than thymus cells + A20 + IL-2 group(57.8%).
(3) Lymphocyte transformation test:thymocytes + mCD99L2~-A20 + IL-2 coculture group had stronger lymphocyte transformation capacity than the group of lack of IL-2, there was a significant difference(P = 0.037).In contrast,thymocytes + mCD99L2~-A20 + TNF coculture group had weaker lymphocyte transformation capacity than the group lack of TNF,there was a significant difference(P = 0.044).
(4) Tumor formation test of animals inoculated subcutaneously with coculture cells: inoculated subcutaneously BALB / c mice with thymocytes + mCD99L2~-A20 coculture cells(experimental group) and thymocytes + A20 coculture cells(control group) which were cultured 1 week respectively.Each group contained 3 mice.3 mice of the experimental group all got tumorigenicity after 2 weeks.But tumorigenicity phenomenon did not appear in the control group;inoculated subcutaneously BALB / c mice with thymus cells + mCD99L2~-A20/A20 + IL-2(experimental group / control group) which were cultured 24h respectively.Each group contained 3 mice.After 2 weeks none of 6 mice got tumorigenicity.Thymus were observed of the experimental group.The size of the thymus was less than the control group.Weight was also less than the control group,the difference was significant(P = 0.017).HE stained sections showed thymocytes were sparse in experimental group.The control group thymocytes cells were dense,liking normal thymus.
(5) Flow cytometry detection of blood of BALB / c mice which received subcutaneous injection of coculture cells:CD4,CD25 and CD8 expression detection of blood two weeks after inoculation.CD4~+ CD25~+ and CD8~+ cell ratios were higher in the blood of mice injected spleen cells + mCD99L2~-A20 coculture cells(3.3%and 8.9%respectively) than the mice injected mCD99L2~-A20 cells only(3.1%and 5.2%). (6) IFN-γand IL-10 detection of coculture supernatant:IFN-γand IL-10 were detcted respectively at 24h,60h and 5d in the supernatant of thymocytes + mCD99L2~-A20 co-culture group(experimental group) and A20 + thymus cells cocultured(control group).The control group contained more IFN-γand IL-10 at 24h and 60h,the difference was significant(P= 0.000,P= 0.000).The experimental group supematant contained more IFN-γand IL-10 at 5d,the difference was significant(P = 0.020).
3.Exploring the relationship of H / RS cells and T cells by chip data analysis and comparing with the mCD99L2~-A20 protein chip
(1) There were 518 difference gene expression by analysing gene expression profiles GSE8388 data of cHL cell line L428 and the B-cell lymphoma cell line Raji.KEGG pathway analysis of genes differentially expressed revealed that cytokines and cytokine receptor interaction(Cytokine-cytokine receptor interaction) pathway and JAK tyrosine kinase and signal transducer and activator of transcription(JAK-STAT) pathway were the most significant pathways of the different genes.Immune biology analysis of different genes showed that there were four significant difference genes, CCL5、CCL22、CCL17 and IL-6.They were closely related to the H / RS cells raising T cells.The expression of them were higher in cHL than B-cell lymphoma.The biological process analysis of different genes showed that IL-6 played a significant role in T cell activation and cytokine synthesis.
(2) There were 302 significant gene differentially expressed by analysing gene expression profiles GSE12453 data of cHL cells and B cell lymphoma DLBCL(Diffuse large B cell lymphoma) cells.There were 363 significant gene differentially expressed between cHL cells and normal lymphocytes.By cross-comparing two groups of different genes we found some specific genes in relation to the pathogenesis of cHL.A total of 48,6 up and 42 down,including a kind of cytokine,IL-16.
(3) Taking use of STRING 8.0 online platform to analyse relationship between proteins which come from the most prominent 14 different cytokines of the HL and B cell lymphoma DLBCL of GSE12453.Compared with pre-made cytokine proten chip we selected six cytokines and chemokines related most closely with the T cells in H / RS background,including RANTES,MIG、CCL22、CCL17、IL-6 and CXCL16.MIG was in the center of their interaction network.
Conclusion
1.H / RS like cells-mCD99L2~-A20 cells got a low tumorigenicity rate injected subcutaneously into normal BALB / c mouse.Tumorigenicity has been greatly improved by radiating immunosuppression,suggested that T lymphocyte immunity as well as change of T lymphocyte subtypes play important roles in the tumorigenicity.Reducing the cytotoxic T cells can relatively increase helper T lymphocyte which is more beneficial to mediate immune tolerance of H / RS cells.Thus tumor formation rate can be improved.
2.Coculture mCD99L2~-A20 cells and T cells to imitate H / RS cell microenvironment in vitro.Explore the interactions of H/RS cell and T cells.Many CD4~+ CD25 ~+Treg cells can be collected by mCD99L2~-A20 cells.The Treg cells can mediate immune tolerance.They can supress the killing effect of CD4~+ Th1 and CD8 ~+ CTL.Th1,Th2 and CTL maintain steady-state environment of tumor cells through some cytokines,such as IL-2、TNF、IFN-γand IL-10.
3.Analyse molecular biological mechanisms of interaction of H / RS cell and background T cells through bioinformatics resources and public chip data analysis software.There are some cytokines and chemokines that are related closely to H / RS cell and background T cells,such as RANTES、MIG、CCL22、CCL17、CXCL16、IL-6、IL-16 and so on.The H / RS like cells mCD99L2~-A20 cell model and the BALB / c mice tumor model constructed by our research group are similar to cHL.
引文
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