Screening Of Cell Membrane Antigens In Human Esophageal Carcinoma Eca-109 Cell Line

Objective:Tumor vaccine is to input antigen to the body directly or indirectly, which stimulates the body's immune system to produce antitumor immune response. Since most of the tumor cell antigens are cell membrane proteins, it is important to collect them for searching tumor antigens. In order to investigate the efficient cell membrane antigens, the techniques such as the modified Neville method were used to collect the cell membrane 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:Since dendritic cells (DC) have the capability for presenting antigen effectively and activating naive T lymphocytes, cytotoxic T lymphocytes (CTL) are the main effector cells in specific antitumor immune response, and helper T cells (T_H) are indispensable in this response. These three above-mentioned cells were purified and then specific antitumor immune responses were performed complemented with cell membrane antigens gained by ultrafiltration. According to the degrees of the responses, the rough scoup of high performance tumor antigens might be screened out.1. The cell membrane proteins of Eca-109 were isolated. First, the cytomembrane was isolated using the modified Neville method (pH 6.3). Second, the proteins were dissolvedout from the cytomembrane with the detergents of Triton X-100 and Octyl- # -D-glucopyranoside respectively.2. The density of cell membrane proteins was measured. Since Triton X-100 could absorb the light under the wavelength of 280nm, the modified Bradford method was carried out to measure the density of various proteins containing detergent. The linear equation of proteins under 595nm wavelength was performed. Then, the density of the cell membrane proteins collected was deduced and the appropriate detergent: protein ratios,including 1:1, 2:1, 6:1 and 10:1, were compared.3. The cell membrane proteins were divided into 6 groups by ultrafiltration. In the groups with Triton X-100 treatment, the molecular qualities of the cell membrane proteins were less than 3 KDa, 3¹0 KDa, less than 10 KDa and 10³0 KDa. In the groups with Octyl-P-D-glucopyranoside treatment, they were 30¹00 KDa and more than 100 KDa. The density of cell membrane proteins was measured in the 6 groups.4. The typing of human leukocyte antigen (HLA) -A,-B and -DRB characteristics of Eca-109 cell were performed and that of the healthy adult volunteers were screened. For typing, the sequence specific primer polymerase chain reaction (PCR-SSP) method used allele-specific primers in the amplifications of 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 alleles were screened to match at least one of the HLA alleles of Eca-109 cell.5. The screened volunteer's DC were gained in vitro. The volunteer's peripheral blood mononuclear cells (PBMC) were isolated with the method of density gradient centrifugation and were cultivated in RPMI-1640 culture medium supplemented with granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-4 (IL-4) and the volunteer's serum. With appropriate cell density, the PBMC were cultured in 24 well culture plate. On the forth day of culture, according to a determinate proportion, the cell membrane proteins and hTNF- a were added to maturate DC. On the twentith day, suspending mature DC were collected.6. The screened volunteer's T lymphocytes were purified in vitro. With flow cytometry (FCM), 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 we...