| Objective: To detect the specific antineoplastic immunological effect in vitro of cytotoxic T lymphocyte(CTL) induced by human dendritic cell loaded with caner/testis antigen NY-ESO-1 peptides, then to obtain the NY-ESO-1 peptide which can induce CTL to kill several kinds of tumor cells powerfully. The studies can provide important theoretical dasis for tumor immunotherapy.Methods:1. Predict NY-ESO-1 antigen epitopes through the methods including hyper-motif, hapto-polynomial and quantized motif, the former is provided by the website of SYFPEITHI(http://www.syfpeithi.de). And we can obtain three HLA-A2 restriction peptides and then synthesized them artificially.2. Routinely cultivate human hepatoma carcinoma cell strain MHCC97-H, colon carcinoma cell strain SW480, brain gliocythma cell strain U251 and gastric carcinoma cell strain SGC-7901. The HLA-A2 phaenotype of the former three kinds of tumor cell strains are all positive, but HLA-A2 phaenotype of SGC-7901 is negative.3. Detect the expression states of NY-ESO-1 in the above tumor cell strains through the methods of RT-polymerase chain reaction(RT-PCR) and immunocytochemical technique, then induce the tumor cell strains without the expression of NY-ESO-1 to renewedly express NY-ESO-1 with the utilization of 5-Aza-2'-deoxycytidine(5-Aza-CdR).4. Induce and cultivate DC of the cord blood of human being with recombination human granulocyte/macrophage colony-stimulating factor(rhGM-CSF) and recombination human interleukin-4(rhIL-4), and then induce DC to be mature with recombination human Tumor necrosis factor-α(rhTNF-α). Observe DC morphous with inverted microscope, ordinary optics microscope and transmission electron microscope, and detect the expression state of DC surface molecules including HLA-DR, CD83 and CD86 with flow cytometry, and identify the function of DC with allogeneic mixed lymphocyte reaction.5. Cultivate T-lymphocytes of the cord blood of human being with recombination human interleukin-2(rhIL-2) and phytohaemagglutinin(PHA), and sensitized DC with NY-ESO-1 peptides, then induce the amplification of CTL with DC. Detect the killing rate of CTL against the tumor cells with the expression of NY-ESO-1 using the release assay of lactate dehydrogenase(LDH), so as to screen out the peptide of NY-ESO-1 which can induce CTL to kill several kinds of tumor cells powerfully.Results:1. Obtained five nonapeptides of NY-ESO-1 whose score were more than 20 through the method of hyper-motif, then selected three nonapeptides including p86-94(RLLEFYLAM), p108-116(SLAQDAPPL) and p159-167(LMWITQCFL) whose binding coefficients were highest through the methods of hapto-polynomial and quantized motif. Then get the peptides by artificial synthesis using chemical synthesis.2. Successfully screened out human gastric carcinoma cell strain SGC-7901 with the expression of NY-ESO-1, but human hepatoma carcinoma cell strain MHCC97-H, colon carcinoma cell strain SW480 and brain gliocythma cell strain U251 were all without the expression of NY-ESO-1.3. The cell strains without the expression of NY-ESO-1 including human hepatoma carcinoma cell strain MHCC97-H, colon carcinoma cell strain SW480 and brain gliocythma cell strain U251 successfully renewedly expressed NY-ESO-1 with the induction of 5-Aza-CdR.4. Successfully cultivated abundant DCs from the cord blood of human being with the united utilization of the cell factors of rhGM-CSF, rhIL-4 and rhTNF-α. After 8 days, the cell volume enlarged, and the cells were semi-adherent and had irregular shape with typical dendritic processes. Then through flow cytometry, the rate of expression of cell surface molecules including HLA-DR, CD83 and CD86 of immature DC and mature DC were respectively 37.12%, 3.77%, 21.58% and 93.53%, 55.21%, 69.12%. It indicates that mDC can stimulation the amplification of lymphocytes.5. Through the release assay of LDH, the killing rates of CTL induced by DC loading with NY-ESO-1p86-94 peptide against human hepatoma carcinoma cell strain MHCC97-H, colon carcinoma cell strain SW480 and brain gliocythma cell strain U251 were 42.41%±2.92%, 32.27%±2.59%, and 36.59%±2.14%, which were significantly higher than the killing rates of the control groups without peptide(P<0.05).Conclusion:1. The antigen epitopes of NY-ESO-1 can be predicted with the united utilization of the methods including hyper-motif, hapto-polynomial and quantized motif.2. 5-Aza-CdR can induce the tumor cell strains without the expression of NY-ESO-1 to renewedly express NY-ESO-1 in vitro.3. DC loading with the HLA-A2 restriction peptide of NY-ESO-1 can effectively promote the amplification of CTL in vitro and induce its specific immune reaction of killing the tumor cells with HLA-A2 phaenotype and the expression of NY-ESO-1.4. Successfully obtained one peptide of NY-ESO-1: p86-94(RLLEFYLAM), which can induce CTL to kill human hepatoma carcinoma, colon carcinoma and brain gliocythma powerfully.
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