| 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,Bglâ…¡and EcoRâ… were 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,NucleofectorTMtransfection 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 Bglâ…¡and EcoRâ… ,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 lipofectamineTM2000,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 LipofectamineTM2000-mediated,24-48 h later,there was little green fluorescence seen only in individual cells under fluorescence microscopy.(2)NucleofectorTMtransfectionA 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 NucleofectorTMtransfection 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 NucleofectorTMtransfection.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.
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