| It has been demonstrated that lack of costimulatory signal is a important reasonfor tumor cells escaping from immunological surveillance. The activation of antineoplasticimmunity of immunifactions depends on the antigen of tumour cells being identified byimmune system. However, because of poor immunogenicity of tumor and somedefect in the process of antigen presentation, tumour could escape from theimmunological recognition. So, the antigenicity of tumor cells and the abilities toidentify and present tumor antigen of antibodies could be enhanced and the body'santi-tumor immunopotency could be strengthened when the tumor vaccine is prepared byintroducting immunity related genes into tumour cells.CD80 is a glycosidoprotein expressing on activated B cells, macrophages,dendritic cells, T cells and cell natural killer cells with antigen presentationcompetence which has costimulate effect in T cell activation process when bindingwith its CD28 ligand. However, recent researches have showed that CD80 is notexpressed onto the surface of tumor cells except minority B lymphoma. Those tumorcells without CD80 expressing could not efficiently induce T cell's activation or even induce specific anergy even if they have immunogenicity. Cells with CD80expressing have immunogenicity. When they are identified by the CTL cells ofprosoma of CD8+T, the first signal could be provided by MHC-I molecularcompounds of tumor antigens and the second signal could be provided by CD80through bingding with CD28 on the surface of T cells. And then, after CD8+T cellshave recognized the double signals, they could be activated and excrete some cellfactors as IL-2 cytokine and then proliferate and differentiate to effective CTL andtheir following dissolving cells abilities could kill tumor cells. The tumor cells withspontaneous extinct could express CD80 and bind with CD28 and then induceanti-tumor immunity. Accordingly, tumour cells introducted with CD80 expressed itcould have immunogenicity and can effectively stimulate anti-tumor immunity, whichcan removal the tumorgenesis of them and can reject the remote same tumor cellswithout CD80 expression by inducing pantosomatous specific anti-tumor immuneresponse.However, there have some limitations in traditionary gene transduction by whichCD80 is directly introducted into the target tumour cells. Firstly, the home range andaction in the recipient's body of the exogenous virus vectors used in these transduction areuncontrolled and probably bring about adverse effect. Secondly, the efficient of these methodsis low because of the limitation of the physiological microenvironment in the recipient. Thirdly,the process is very complicated and time consuming during the gene expression. Fourthly,exogenous genes are prone to degrade and the expression are unstable when integrated in thechromosome of the host cells unclear which lead to the difficulty to apply these methods in theclinic use. So, Along with the deeply recognize of GPI anchoring signal, scientists beginto anchor the protein on the cell surface by GPI anchor to play their role of tumorvaccine.GPI anchoring protein anchor on the cells surface by GPI architecture at the C-terminal without transmembrane domain and intra-cellular domain and notspanning cellular membrane lipid bimolecular leaflet. When GPI anchoring proteinsco-incubated with cells, they can automatically cohered to cellular membrane andretain their conjugated capability with their natural ligand. Using above-mentionedcharacteristic of GPI and anti-tumor functions of CD80, we are going to generecombine with the peptide exon at CD58 C-terminal and the ecto-exon of CD80(peptide exon at CD58 C-terminal hasωsite and the expression product was GPIanchoring protein), then to express the recombined gene in the eukaryotic expressionsystem and obtain GPI anchoring CD80 after purification. CD80 could anchoring onthe tumour cell surface by GPI's specific structure and its intrinsical costimulatefunction retains. This kind of tumor vaccine has higher performance and safety andstability and lower time consumption than the vaccine through traditionary genetransfection. So, GPI-CD80 was a new safe generation vaccine with highperformance and offer a firenew way for immunotherapy on tumors.Objectives1. To express pRM10/GPI-CD80 eukaryotic expression plasmid in CHO cellsand select CHO cells stably expressing GPI-CD80 fusion protein.2. To extract the cellular membrane protein with stable GPI-CD80 expressingand purify the GPI-CD80 proteins by immunoaffinity chromatography and thenidentificate them.3. To study the antitumor effects of GPI-CD80 fusion protein and itsmechanisms.Methods1. pRM10 and pRM10/GPI-CD80 were differently transfected into CHO cells through LipofectamineTM 2000 and then transfected cells were selected in G418selective medium and those CHO cells stably expressing GPI-CD80 were obtained.Then, the efficacy of protein GPI-CD80 expressing on the surface of cell membranewas detected by flow cytometry and and identified by immunofluorescence.2. GPI-CD80 membrane protein was purified by affinity column which preparedwith CNBr-Sepharose 4B and mouse anti-homo CD80 monoclonal antibody After thecellular membrane protein of CHO cells with stable GPI-CD80 expressing wereextracted. Then, GPI-CD80 fusion protein was identified by Western-Blot and theexpression of CD80 on the CHO cells surface of before and after PIPLC treatmentwas detected by flowcytometry to judge whether or not the anchoring form was GPIanchoring. Then, to detect the stability of anchoring of GPI-CD80, HepG2 cells andGPI-CD80 protein were co-cultured and the rate of positive cells was detected byfiowcytometry when HepG2 cells co-cultured with GPI-CD80 fusion protein fordifferent time.3. Tumor vaccine was prepared by co-culturing purified GPI-CD80 with HepG2cells and being inactivated by Mitomycin of and HepG2 cells inactivated byMitomycin was the control group. Two groups of vaccines were co-cultured withmice splenic lymphocyte, then the changes of lymphocyte proliferation at 0 h, 24h, 48h, 72 h and quantity of IL-2, IFN-γwere detected by MTT-assay. The activity of CTLwas detected by LDH. The changes of gross tumor volume were measured every 3days after different vaccines were implanted on the nude mice bearing cancer withHEPG2 cells 4 days after HepG2 cells were inoculated in nude mice. PBS used asblank control group and HepG2 cells inactivated by Mitomycin ascontrol group.4. Statistical analysis: statistical analysis was executed on the SPSS11.5. Thecomparison of IL-2,IFN-γ,CTL levels between two groups based onIndependent-Samples T Test. The comparison of T leukomonocyte's OD value and gross tumor volume among different groups and times based on ANOVA for repeated measures. If P＜0.0,bilateral LSD (when mean square was regularity ) or Tarnbane' s T2 (when mean square wasnot regularity ) was executed between the groups and times.Results1. CHO cells would die at different degree at different G418 levels. When theG418 at 800μg/ml,900μg/ml,1000μg/ml levels CHO cells could be killed thoroughlyat the fourteenth day culture, and when the G418 at400μg/ml,500μg/ml,600μg/ml,700μg/ml levels CHO cells could not be killed thoroughly. So wedecided that 800μg/ml level was G418 selecting level of CHO cells at 10％serumconcentration.pRM10/GPI-CD80 was transfected into CHO cells through lipofectamine andthe positive clones were obtained by G418 selecting. The intensive fluorescenceexpressed on the surface of cells membrane was detected by flowcytometry andimmunofluorescence, and fluorescence on the surface of CHO cells transfected withpRM10 could not be detected, which meant that GPI-CD80 was anchoring on thecells membrane. The positive rates and mean fluorecence intensity of GPI-CD80espressed on CHO cells surfaces expressing CD80 was as high as 90.02％, 42respectively, while corresponding values of control group was as low as 0.50％, 3respectively, and those of CHO cells transfected with pRM10 was 8.95％, 10respectively.2. Purified GPI-CD80 fusion protein was obtained through immunoaffinitychromatography. Protein brown straps appeared near 60KD by Western-Blot. TheCHO cells stable expressed GPI-CD80, CHO cells and CHO cells transfected withpRM10 were trypsinizated before resuspened with PBS, then mice-homo FITC-CD80antibody was added to it, and the positive rates and mean fluorecence intensity of CD80 expressed on cell surfaces were be detected. The results showed that thepositive rates and mean fluorescence intensity of CHO cells was 0.47％,2.77respectively, the CHO cells transfected with pRM10 was 7.62％,9.47 respectively andthe CHO cells stable expressed GPI-CD80 was 90.02％,42 respectively. After the cellgroups above-mentioned were treated by PIPLC, the positive rates and meanfluorescence intensity cells of two control groups showed hardly changes, while thevalues of CHO cells stable expressed GPI-CD80 changed to 6.34％,7.72 respectively.The results showed that the protein expressed on CHO cells surface was GPIanchoring.After GPI-CD80 fusion protein and HepG2 cells being co-incubated for 30 min,2 h, 4 h, 8 h, 16 h, the positive rates of CD80 fluorescence showed on surface of cellswere 78.02％, 75.46％, 78.29％, 80.40％, 82.12％respectively detected byflowcytometry. There was no significant difference among the changes of positiverates of different samples. It showed that GPI-CD80 fusion protein could anchoronthe cellular membrane after GPI-CD80 and HepG2 cells co-incubated, and thisanchoring was fair stable.3. The OD values of mice splenic lymphocyte rose after stimulated by tumorvaccine, which at 24 h, 48 h,72 h were 0.44±0.14,0.66±0.03 and 0.99±0.27respectively, there were significant difference between control group and experimentgroup (F=63.958, P=0.000), and among different time points (F=51.912, P=0.003). The quantity of IL-2,IFN-γwere detected in the cell culture fluid of differentgroups, and the value of experiment group were 115.94±3.57, 261.96±19.06,significantly higher than those of control group (P=0.000). CTL of spleniclymphocyte were significantly higher than those of control groups (t=11.37,P=0.000) in the detection of CTL activity after leukomonocyte stimulated by tumorvaccine. There were significant difference among the value of gross tumor volumebetween different groups after different vaccines were implanted on the nude micebearing cancer (F=801.801, P=0.000), which were in blank group, control group,positive group from smaller to larger.Conclusions1. pRM10/GPI-CD80 was expressed into CHO cells successfully and the CHOcells stable expressing GPI-CD80 were obtained by G418 selection.2. The GPI-CD80 fusion protein we obtained could stably anchor on the cellsmembrane.3. The competent GPI-CD80 fusion protein could be obtained by immunoaffinitypurification.4. GPI-CD80 fusion protein could inhibit the growth of tumor on nude micebearing cancer, which might be due to its inducing the proliferation of lymphocytes,stimulating the secretion of cytokine IL-2,IFN-γand enhancing the activity of CTL. |
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