Role Of LAT In CD59 - Mediated T Cell Activation Signal Transduction In T - Line Leukemia Nude Mice

Objective To establish lymphocytic leukemia animal model with Jurkat cell in nude mice and identifying, research the effect of LAT and palmitoylation in T lymphocytic activation and signal transduction though GPI-anchored protein CD59.Methods Cultured Jurkat cells, LAT-EGFP, LAT-M-EGFP fusion proteins constructed and carried by lentivirus vectors, then transfected into Jurkat cells to establish stably expressing cell lines which were named LAT group, LAT-M group. The five weeks old female BALB/C mice were randomly divided into four groups, including three experimental groups: normal Jurkat group, LAT group, LAT-M group, and one blank control group, each group had six. After two consecutive days of intraperitoneal injection of cyclophosphamide, experimental mice tail intravenous Jurkat cell lines 5 x 106 / one and control group mice tail intravenous injection amount of PBS solution in two days.Then observed the symptoms of mice, weight and the number of WBC in peripheral blood at the first, the second, the third and the forth week. Take pathological tissues in HE staining. ELISA was used to test the level of interleukia(IL)-2 in serum. Tested positive rate of tumor cells through FCM. Detected the expression of Bcl-2, and Caspase-3 in liver tissues of model mice in immunohistochemistry methods. Laser scanning confocal microscope was used to observe the location of Lat before or after CD59 antibody cross-linking.Results After vaccinating tumor cells, experimental mice gradually appeared symptoms which like weight loss, camponotus, listlessness and so on, weight had decreased, WBC count in peripheral blood had increased and the survival time was shortened. The tumor cells had seen in HE staining for liver and spleen tissues and in marrow. The mice of blank control group were all alive, These meant the animal model had established successfully. In experimental groups, symptoms in mice with LAT group was more obvious, they had been in articulo mortis and cachexia in the forth week. LAT group had the highest WBC count and the least survival time. The results of ELISA had shown that LAT group had the highest rate of IL-2 than other groups. The levels of Bcl-2 in Jurkat and LAT-M group were lower than LAT group. The expression levels of Caspase-3 in LAT-M group were higher than LAT group and Jurkat group. The positive rate of tumor cells through FCM shown that LAT group was the highest. The immunofluorescence results showed that in LAT group, LAT molecules distributed on the membrane and somehad clustered in lipid raft. After cross-linked CD59, the clusters were more obvious. In LAT-M group, the LAT molecules scattered on the membrane and couldn’t clustered.Conclusion The research had successfully established the animal model of human Jurkat cells in nude mice. LAT and palmitoylation play a positive role in the proliferation and activation of T cell, CD59 can modulate T cell signal transduction through providing palmitoylation group for LAT.