| Objective:Tumor vaccine is to input antigen to the body directly or indirectly. This antigen stimulates the body's immune system to produce antitumor immune response. Since most of the tumor cell antigens are cytomembrane proteins, it is important to collect them for searching tumor antigens. In order to investigate the efficient cytomembrane antigens, the techniques such as the modified Neville method and mild acid wash method were used to collect the cytomembrane proteins with different molecular weights in human esophageal carcinoma Eca-109 cell. To broaden the clinical applicability of antigen-based immunotherapy in human esophageal carcinoma, tumor antigens were screened and the rough scoup of high performance tumor antigens may be screened out.Methods:l.The cytomembrane proteins of Eca-109 were isolated. First, the cell membrane was isolated using the modified Neville method (pH 6.3). Second, the proteins were dissolved out from the cytomembrane with the detergents of Triton X-100 and Octyl-3-D-glucopyranoside respectively.2.The density of cytomembrane proteins was measured. The modified Bradford method was carried out to measure the density of various proteins containing detergent under 595nm wavelength. The linear equation of proteins was performed. Then, the density of the cytomembrane proteins collected was deduced and the appropriate detergent: protein ratios were compared.3.The cytomembrane proteins were divided into 6 groups by ultrafiltration. Cytomembrane proteins with Triton X-100 treatment, were divided into less than 3KD,3~ 10KD, less than 10KD and 10~30KD four groups by molecular weight. Those with Octyl-B-D-glucopyranoside treatment were divided into 30~100KD and more than 100KD two groups. The density of cytomembrane proteins was measured in the 6 groups.4.The typing of HLA (human leukocyte antigen) -A, -B and -DRB of Eca-109 cell were performed and that of the healthy adult volunteers were screened. For typing, the PCR-SSP (sequence specific primer polymerase chain reaction) method used allele-specific primers for PCR. The amplified DNA was determined using agarose gel electrophoresis and the HLA alleles of Eca-109 cell were identified. With serologic HLA typing technology, the healthy adult volunteers' HLA gene locus alleles were screened to match at least one of the class I HLA gene locus alleles of Eca-109 cell.5.The screened volunteer's DC were gained in vitro. The volunteer's PBMC (peripheral blood mononuclear cells) were isolated with the method of density gradient centrifugation and were cultivated in RPMI 1640 culture medium supplemented with GM-CSF, IL-4 and autologous serum. With appropriate cell density, the PBMC were cultured in 24-well culture plate. On the 4th day of culture, according to a determined proportion, the cytomembrane proteins were added to immature DC. On the 6th day TNF- a was added to maturate DC. On the 7th day suspending mature DC were collected.Cryopreservation of DC: Antigen-preloaded, mature (day 7) DC were frozen at 1 癈/min in pure autologous serum+10% DMSO+5% glucose at a cell density of 10 X 106/ml.6.The screened volunteer's T lymphocytes were purified in vitro. With FCM (flow cytometry), the volunteer's PBMC co-cultivated with IL-2 were observed to get their differentiation information. Then, the volunteer's PBMC were re-collected and were stimulated with PHA. According to the detection result of FCM, co-cultivated with IL-2 for four weeks, the purified T lymphocytes were collected.7.By MTT reduction assay, specific cytotoxicity of CTL against Eca-109 cells was analyzed. The purified T lymphocytes were used as effector cells and Eca-109 cells were used as target cells. The effector : target ratios were 8:1,16:1 and 32:1. The mature DC loaded with antigens were added. Meanwhile, the positive, negative and effective controll groups were set up. Each group had triplicate wells. Then thespecific cytotoxicity of CTL was analyzed in 96 well culture plate.8.Mild acid wash method: The flasks (250ml) of confluent monolayer cultures of...
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