| BackgroundMalignant 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.Objective1.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.Method1.Inducing tumor formation of mCD99L2~-A20 cells by changing T cell subsets of BALB / c miceInoculating 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 cocultureCoculturing 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 chipTaking 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.Result1.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.Conclusion1.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.
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