Mechanism Of Inhibition On Parasitophorous Vacuole Fusion With Lysosome By PRU Toxoplasma Gondii

Objectives:To establish a model that PRU-T2 infected macrophages in vitro. Then the dynamics of fusion protein( Rab5, Rab7, EEA– 1)which mediate parasitophorous vacuole fusion with lysosome, were detected by fluorescence technology.To explore the mechanism of inhibition on parasitophorous vacuole fusion with lysosome by Toxoplasma gondii. To provide evidence for reasonablely designing vaccine by exploring the mechanism of toxoplasma gondii immune escape.Methods:The model of cells infected by toxoplasma was established in vitro. Intra-abdominal macrophage in mice, mice spleen macrophage, mice alveolar macrophage, RAW264.7 cell line were cultured. Cells were infected by PRU - T2 Toxoplasma (heat-killed PRU-T2 as negative control).On the point of 30 min, 60 min, 90 min and 120 min, coerslips were taken out, fixed and stained. The time and quantity of parasitophorous vacuole formed were compared. Appropriate cells were chosen for further research. The dynamic distribution of Rab5,Rab7 and EEA -1 on parasitophorous vacuole membrane was detected by indirect immunofluorescence fluorescence technology. Logarithm growth period cells were digested and blown. The cells were cultured in 24 hole board which were presented coverlips.The macrophages were infected by toxoplasma gondii after sticking wall. The coverlip was taken out, kept fixing with 4% polyphosphate formaldehyde for 10-30min. The coverlip was processed in 0.5% Triton-100 for 10-20min.Then 2%BSA was added. The coverlip was incubated for 1h in 37℃incubator. First antibody working liquid was added to it,4℃overnight, washed. Second antibody working liquid was added to it.And then the coverlip was incubated for 1h in 37℃incubator again,washed, dried in the air, camera shooting.Results:1 The state of the four kinds of cell which invaded by PRU - T2 toxoplasma besides the time and quantity of parasitophorous vacuole formed were compared, RAW264.7 cell line was suitable for this study. PRU - T2 removed from vaccination rat celiac was used to infect intra-abdominal maerophage in mice, mice spleen macrophages, mice alveolar cavity macrophages, RAW264.7 cell line. At the point of 30min,the parasitophorous vacuoles of 50 cells, were counted, respectively were 50,10-50,50-80,50-100.2 Rab5 was persisiting on the membrane of parasitophorous vacuole while the content of Rab5 on the membrane of phagosome with heat-killed toxoplasma was declining until disappeared. Rab7 couldn,t be detected on the membrane of parasitophorous vacuole.The content of Rab7 on the membrane of phagosome with heat-killed toxoplasma was rising. EEA-1 couldn,t be detected on the membrane of parasitophorous vacuole either while could be detected on the membrane of phagosome with heat-killed toxoplasma.Conclusions:1 There were more and earlier parasitophorous vacuole formed in RAW264.7 cell after being invaded by toxoplasma.RAW264.7 cell was more easily cultured,which was suitable for further research.2 The dynamic changes of Rab5,Rab7,EEA-1 on the membrane of parasitophorous vacuole suggested that Rab5 should be persisted on the membrane of parasitophorous vacuole, parasitophorous vacuole should be stopped at early endosome stage by excluding Rab7,and EEA-1 should be refused by the membrane of parasitophorous vacuole,which result in the inhibition on parasitophorous vacuole fusion with lysosome and contribute to the success of immune escape.