| BackgroudLymphomas are immune system malignant neoplasms of cells native to lymphoid tissue.Along with the wide use of immunosuppressant for organ transplant, AIDS occurence,and chemotherapy for tumors,the occurrences of lymphomas have drastically increased in recent years.Different from non-Hodgkin's lymphoma(NHL),Hodgkin's lymphoma(HL) is a peculiar form of malignancy in which the clonal B cell population,the so called Hodgkin/Reed-Sternberg(H/RS) cells and their variants,is responsible for less than 1%of the bulk of the tumor.The mechanism of development of HL and H/RS cell is still illusive.The relationship between H/RS cell and background cells is also restricted for lack of suitable animal model which mimics the characteristics of HL. There are reports that human H/RS cell is formed for the low expression of CD99 (MIC2).Our research work have previously transfected the mouse B lymphoma cell line A20,which expressed mouse CD99 antigen-like 2 gene,using Letivirus ShRNA vector and constructed subseries of A20 cell line with low mCD99L2 gene expression,named "LV-mCD99L2-A20".Among the LV-mCD99L2-A20 cells,giant cells like human HL H/RS cells were found.Whether these giant cells could consistently present during continuous passage culture of LV-mCD99L2-A20 cells and what are the immunophenotypes of these cells? How about the tumors induced by LV-mCD99L2-A20 cells in the nude mouse and BALB/c mouse? What's the difference between LV-mCD99L2-A20 cells and A20 cells in mouse with nomal immune system?These problems need to be investigated.The animal model people often used were engrafted immunodeficiency animals inoculated with lymphoma cell lines or the tissue from lymphoma patients,which were of great importance during the research into molecular mechanisms of lmphomas.Most of them are immunodeficiency animals.Lymphoma is a kind of tumor envolved with body immune system.For lack of T lymphocyte or/and B lymphocyte,neither the nude mouse nor the severe combined immunodeficiency mouse could mimic the immune enviroment of human body and it is hard to use these models to reveal the interaction between lymphoma and immune system of human body.Therefore these animal models are limited in the research of clinic therpy and drug usage.The abundant reactive cells in HL were related not only with whole immune system of the body,but the immuno-microenviroment induced by H/RS cells.It has been postulated that the presence of abundant reactive cells in HL tissue is due to the production of several kinds of cytokines and/or chemokines.Really what is the relationship between HL H/RS cell and cytokines? Is there any difference in the cytokines' expression between A20 cells and LV-mCD99L2-A20 cells? What kinds of cytokines will be induced in BALB/c mouse by A20 cells and LV-mCD99L2-A20 cells respectively? Will the secretion of cytokines in LV-mCD99L2-A20 cells and tumor tissues benefit the construction of real HL animal model? After the costruction of A20/LV-mCD99L2-A20 animal model,our work will discuss the relationship between LV-mCD99L2-A20 cell model and/or animal model and costruction of HL.Objective1.Identifying the subseries which express low level of mouse CD99 antigen-like 2 gene(mCD99L2) of mouse B lymphoma cell line(A20),named LV-mCD99L2-A20, so as to investigate the importance of mCD99L2 in the formation of Hodgkin/ Reed-Sternberg(H/RS) like cell.2.Inoculating mouse B lymohoma cell line A20 to homologic BALB/c mouse in different ways,so as to construct mouse B lymphoma animal models and compare the difference between them,thus providing suitable animal models for research in B cell lymphoma.3.Using LV-mCD99L2-A20 cells to inoculate BALB/c mouse,investigating the tumor formation,observing its pathological characteristics,identifying its molecular biology and detecting its immunophenotypes so as to compare the biological difference between in vivo and in vitro,comparing the pathological characteristics between LV-mCD99L2-A20 cell-induced tumors and A20 cell-induced tumors,thus analyzing the relationship between the difference and human HL.4.Applying the software of bioinformatics to retrieve and analyze the relationship between HL and cytokines.Using mouse cytokine antibody arrays to investigate the expression of cytokine in the cells and tumor tissues,comparing its difference and analyzing the relationship between the diffenrence and construction of human HL like animal model,thus providing experimental data for our profound research work.Method1.During the period of continuous passage culture of A20 cells formerly transfected with LentiVirus-mCD99L2 vector,named LV-mCD99L2-A20 cells,choosing cells of different passages respectively:examining the integrated status of vector using DNA-PCR;analyzing RNA interference efficiency by RT-PCR and real time RT-PCR, observing morphological characteristics and counting numbers of giant cells under light microscope,detecting expression of mouse CD30 molecules in LV-mCD99L2 -A20 cells by immunofluorenscence,observing the growth curve by MTT and detecting the cell cycle and immunophonotypes of A20 cells and LV-mCD99L2-A20 cells such as CD3,CD4,CDS,CD19,CD20,CD30.2.Innoculating mouse B lymohoma cell line A20 to homological BALB/c mouse by subcutaneous inoculation,intravenous injection,spleen inoculation,intraperitoneal injection and transplantation of tissue from nude mouse,observing the tumor formation,paraffin imbedding the organs of animals,pathological section and HE staining to observe its pathological characteristics.Detecting CD3,CD4,CD8,CD19, CD30 expression of the cell suspension of tumor tissues with Fluorescence Activated Cell Sorter(FACS) technology.Comparing the difference of lymphocyte subpopulation in normal mouse and mouse with or without tumors.3.Using identified LV-mCD99L2-A20 cells to inoculate homologic BALB/c mouse by subcutaneous inoculation and intravenous injection,observing the tumor formation,paraffin imbedding the organs of animals,pathological section and HE staining to observe its pathological characteristics,detect the expression of CD3,CD4, CD8,CD19,CD30 in cell suspension of tumor tissues by FACS and also compare the difference of lymphocyte subpopulation in peripheral blood and spleen of normal mouse and mouse without tumors after A20 inoculation and LV-mCD99L2-A20 inoculation.4.Using bioinformatics software Geneclip to retrieve the articles about the HL and/or H/RS cell and 62 kinds of cytokines,analyzing the relationship between them.Using RayBiotech mouse cytokine Antibody Arrays to investigate the expression of cytokines in A20 and LV-mCD99L2-A20 cells and their related tumor tissues, extracting and quantitating proteins,sealing and incubating membranes,detecting expressions of cytokines,imaging photos and analyzing the difference between them.Result1.Identification of subseries of A20 with low expression of mCD99L2 gene (LV-mCD99L2-A20):(1) Giant cells like Hodgkin/Reed-Sternberg cells(H/RS-like cell) were found during continuous passage culture of LV-mCD99L2-A20 cells.The number of giant cells in A,B,C,D passage cells were more than those in A20 cell line,with dramatic difference(P＜0.05)(2) During continuous passages of LV-mCD99L2-A20 cells,282 bp fragments of LV-mCD99L2 ShRNA vector were detected by DNA extraction and following PCR reaction.(3) Expression of mCD99L2 gene mRNA in LV-mCD99L2-A20 cells was lower (about 50%)than those in A20 control group revealed by RT and real-time PCR.(4) The proliferating ability of LV-mCD99L2-A20 cell deteced by wethod of MTT is slower than the contral A20 group and mock group,with dramatic difference (P＜0.05).(5) Cell cycle detection by FACS revealed that S phase of each group is of no significant difference(P＞0.05),while the G2 phase is longer than the contral A20 group and mock group,with significent difference(P＜0.05).(6) LV-mCD99L2-A20 cells,including giant cells,were positive labeled by mouse CD30 antibody in immunofluorenscence test.(7) The CD3 expression of LV-mCD99L2-A20 cell(7.47±1.27%)is lower than A20 cell(18.93±3.87%),CD8 expression of LV-mCD99L2-A20 cell(8.33±3.89 %)is lower than A20 cell(18.27±3.45%),CD20 expression of LV-mCD99L2-A20 cell(13.10±5.16%)is also lower than A20 cell(35.33±2.25%),while CD30 expression of LV-mCD99L2-A20 cell(76.0±12.44%)is higher than A20 cell (41.70±2.60%),all with significent difference(n=3,P＜0.05).The CD4 and CD19 expression of LV-mCD99L2-A20 cell were of no significant difference with A20 cell (P＞0.05).The mock group is of no significant difference with A20 cell in each CD antigen ex[ression(P＞0.05).2.Construction of animal model using mouse B lymphoma A2O cell line using different methods:(1) There are no tumor formed in 2×10~5 BALB/c mouse group,while 100%tumor formation in the BALB/c mouse of subcutaneous inoculation(2×10~6 and 2×10~7) groups and tissue(from nude mouse) trans-plantation group after inoculating about 15.29±3.2 days,7.0±0.82 days and 6.29±0.49 days,respectively. (2) There are 71.4%(5/7) and 100%(7/7) tumor formation in intravenous injection (2×10~6 and 2×10~7) group respectively,71.4%(5/7) in spleen inoculation group and 14.3%(1/7) in intraperitoneal injection group after about 76.8±12.0 days,26.1±7.99 days,32.6±5.99 days and 27 day.The tumor formed in several organs of mouse,such as liver,spleen pancrease,kidney,esophagus,stomach,intestine,mesentery,brain, lymphocytic node,bone,uterus and muscles et al.(3) The tumor cells diffused with large round or irregular nucleus in the tissues, nucleoli is obvious and cytoplasm is light staining,somewhat like diffuse large B cell lymphoma(DLBCL).(4) The expression of CD3,CD4,CD8,CD19,CD30 in the cells from tumor tissues(%) are 49.27±23.75,6.07±3.65,51.2±23.1,67.06±16.39 and 37.93±17.03, respetively.(5) FACS detection of proportions of lymphocyte subsets in peripheral blood show significant differences in CD3 and CD4 expressions between each groups.The proportions of lymphocyte subsets in the blood of mouse with tumor genesis after A20 cell inoculation are lower in CD3(P=0.014) and CD4(P=0.009) expression than those of normal BALB/c mouse and also lower in CD3(P=0.004) and CD4(P=0.006) expression than those in mouse without tumor genesis after A20 cell inoculation.The proportions of lymphocyte subsets in the blood of mouse without tumor genesis after A20 cell inoculation are of no significant difference from those of normal BALB/c mouse:CD3(P=0.592),CD4(P=0.867),CD8(P=0.398),CD19(P=0.510) and CD4/CD8 (P=0.545) ratio.3.Construction of animal model using identified LV-mCD99L2-A20 sbseries:(1) There are 100%tumor formed in the nude mouse of subcutaneous inoculation (2×10~7) group after average about 13.33±4.63 days,while only 7.1%(1/14) tumor formed in the BALB/c subcutaneous inoculation(2×10~7) group and nude tumor tissues transplanting group,after 10 days and 6 days,respectively.Intravenously inoculated BALB/c mouse group(n=28) has no tumor formed till three months after inoculation.(2) Tumor cells in nude mouse and BALB/c mouse indicated different sizes under light microscope,diffuse contribution,large and deep stained nucleus,sparse binucleated,polynucleated observation,which was similar to pathological characteristics of human HL H/RS cells.Besides,lymphocytes infiltrated into tumor tissues of BALB/c mouses,with high expression of CD3+ T lymphocyte,CD4+ T lymphocyte,CD8+ T lymphocyte and high CD30 expression.(3) During primary culture of tumor tissues,282 bp fragments of ShRNA vector were detected in LV-mCD99L2-A20 B1 cells.Expression of mCD99L2 gene mRNA in LV-mCD99L2-A20 B1 cells was lower than A20 control group revealed by RT-PCR.(4) FACS detection of proportions of lymphocyte subsets in peripheral blood show significant differences in CD3,CD4,CD8 and CD19 expressions between each groups.The proportions of lymphocyte subsets in the blood of mouse without tumor genesis after LV- mCD99L2-A20 cells inoculation are of significant differences from those of normal BALB/c mouse--lower in CD3(P=0.014),CD4(P=0.018) and higher in CD19(P=0.012) expression,while are of no significant differences in CD8(P=0.075).The proportions of lymphocyte subsets in the blood of mouse without tumor genesis after LV- mCD99L2-A20 cells inoculation are of significant differences from those of A20 inoculated BALB/c mouse -- lower in CD3(P=0.004), CD4(P=0.012) and CD8(P=0.007) expression while no significant differences in CD19(P=0.052) expression.(5) FACS detection of proportions of lymphocyte subsets in spleen show significant differences between each groups.The proportions of lymphocyte subsets in the spleen of mouse without tumor genesis after LV-mCD99L2-A20 cells inoculation are of significant differences from those of normal BALB/c mouse --lower in CD3 (P=0.000),CD4(P=0.001),CD8(P=0.000) expressions while higher in CD19(P=0.000) expressions and CD4/CD8(P=0.000) ratio.The proportions of lymphocyte subsets in the spleen of mouse without tumor genesis after LV-mCD99L2-A20 cells inoculation are of significant differences from those of A20 inoculated BALB/c mouse - lower in CD3(P=0.009),CD4(P=0.020) expressions,higher in CD19(P=0.020) expression, while no significant differences in CD8(P=0.170) and CD4/CD8(P=0.646) ratio.4.Expression of cytokines in A20 / LV-mCD99L2-A20 cells and tissues:(1) The up-regulated cytokines(≥1.5 folds) in LV-mCD99L2-A20 cells,when compared with A20 cells,include CD30T,IL-12p40/p70,IL-3,IFNγ,,CXCL16, MIP-1αand CD40,among which CD30T is up-regulated by 2.91 folds.There is no down-regulated(≥1.5 folds) cytokines in LV-mCD99L2-A20 cells compared with A20 cells.(2) The up-regulated cytokines(≥1.5 folds) in LV-mCD99L2-A20 cell-induced tumor in BALB/c mouse,compared with those in LV-mCD99L2-A20 cells,include VCAM-1,MIP-1γ,MIG,IL-10,RANTES,PF-4,CXCL16,VEGF,MIP-3αand P-Selectin,among which VCAM-1,MIP-1γ,MIG and IL-10 up-regulated in excess of two folds.The down-regulated cytokines(≥1.5 folds) include IL-6,Eotaxin,TNFα,TIMP-1,SCF,IFNγ,IL-3,CD30 T,IL-5,IGFBP-6,TECK,L-Selectin,Fas Ligand,IL-3 Rb and IL-9,among which IL-6 down-regulated in excess of three folds.(3) When compared with A20 cell-induced tumor,the down-regulated cytokines(≥1.5 folds) include Eotaxin,IGFBP-6,IL-12 p40/p70,IGFBP-5,MIP-1α,KC and CXCL16 in LV-mCD99L2-A20 cell-induced tumor in BALB/c mouse,among which Eotaxin and IGFBP-6 down-regulated in excess of two folds,while the up-regulated cytokines(≥1.5 folds) is only VEGF.Conclusion1.There exist giant cells with morphologic characteristics that like human HL H/RS cells in the subseries of A20 which show low expression of mCD99L2 gene(LV-mCD99L2 -A20 cell).The biological characteristics and immunophotypes of LV-mCD99L2-A20 cell much more mimics HL than A20 cell.2.The subcutaneously transplanted tumors of BALB/c mouse are formed in a shorter time period after A20 cell inoculation than those in other animal models.The hematogenous disseminated animal model by more than 10~6 A20 cell inoculation is involved with several visceral organs.These animal models provide suitable animal models for research of B lymphomas in mouse with nomal immune function.3.LV-mCD99L2-A20,when inoculated into BALB/c mouse,has lower tumor genesis compared with A20 inoculation,while more giant cells with the morphologic characteristics that like human HL H/RS cells dispersed in LV-mCD99L2-A20 cell-induced tumors than in A20-induced tumors in BALB/c mouse,accompanied by some infiltrating lymphocytes.4.LVomCD99L2-A20 cell is more likely to express cytokines usually appeared in HL cell lines or in H/RS cells.There exist differences between the cytokine expressions of LV-mCD99L2-A20 cell-induced tumors and A20-induced tumors in BALB/c mouse.New pointFrom three levels:in vitro investigation,nude mouse and BALB/c mouse in vivo investigation,the research compared the fifferences systematically between A20 and LV-mCD99L2-A20 cell models,between their animal models by morphological observation,detection of immunophonotypes and biological characteristics combining with primary investigation of immune function and cytokines expressions.The results revealed and confirmed that the low expression of mCD99L2 gene is of great importance to the construction of H/RS-like cell model in vitro and in vivo and also revealed a better prognosis of LV-mCD99L2-A20 cells than A20 cells in animal with normal immune function,which may be some similar to genesis of HL. Through primary detection of cytokines in vitro and in vivo,analyzing the relationship between LV-mCD99L2-A20 cell model,animal model and construction of HL animal model.All of these made a better basis for prolonged construction of HL animal model. |
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