Influence Of Regulation Of Host Cell Autophagy On The Proliferation Of Toxoplasma Gondii In Host Cells

Objective: Detect the degree of host cell autophagy infection-induced byToxoplasma gondii to study the role of autophagy in the replication process ofToxoplasma proliferation.Methods: Detect the replication kinetics and morphology of Toxoplasma gondiiinfection at different time points after using autophagy inhibitors, accelerators to judgethe role of autophagy in the replication process of Toxoplasma gondii proliferation.Kunming mice (female,5-7weeks old, weighing16-20g) for the conservation andexperimental RH strain. The mice are infected by intraperitoneal injection of RH strainwith an average onset of4-5days, then extract ascites sterilely and purify Toxoplasmagondii, infect HEF cells after counting for experiments. Experimental groups were asfollows:①normal control group (HEF cells)②RH strain+HEF cells③autophagy inhibitor+RH+HEF cell lines④autophagy inducer+RH strain+HEFcells. Then, these cells were infected by Toxoplasma gondii tachyzoites at given MOI(2:1,4:1,8:1,16:1). Host cell autophagy was detected through acridine orange stainingand MDC (Monodansylcadaverin) fluorescence staining at different time points (1h,2hs,4hs,8hs,24hs,48hs,96hs post infection). Meanwhile, the HEF cells are cultured in sixwell plates after counting, after the cells climb slice and attach to the wall, performGiemsa staining and acridine orange staining respectively, and perform MDC staining in 24-well plates. Detect the condition of HEF cells autophagy with acridine orangefluorescence staining and MDC fluorescence staining, and detect the replication kineticsof Toxoplasma gondii infection at different time points using Giemsa staining. Throughthe establishment of a mouse model of T. gondii infection，using autophagy inhibitors and inducers of autophagy Toxoplasma infection in amouse model intervened respectively. Fluorescence quantitative PCR proliferation ofToxoplasma gondii under different experimental conditions.Results: Observation of Toxoplasma gondii proliferation: Observe theparasitization of Toxoplasma gondii tachyzoites in the cell with Giemsa staining.Examine100cells under microscopy randomly to detect the invasion rate and theaverage number of intracellular proliferation at different time. Invasion rate=thenumber of cell invaded/100×100%; average number of intracellular proliferation=sum of the number of worms in each cells/the number of cell invaded×100%.Acridine orange and MDC fluorescence staining can be used to observe the occurrenceof autophagy process. The results of acridine orange and MDC(Monodansylcadaverin)fluorescence staining showed that autophagy inhibitors and inducers could inhibit andpromote the autophagy of HEF cells respectively. From the results Giemsa staining, itwas found that the proliferation of Toxoplasma gondii in HEF cells could be promotedwith autophagy inducers and be inhibited with autophagy inhibitors. Quantitative PCRresults showed that bafilomycin A1dose and the number of mice in vivo Toxoplasmanegative correlation, while inducing agent lithium chloride and the number of mice invivo Toxoplasma gondii positive correlation.Conclusion：After the infection of Toxoplasma gondii, the autophagy of HEF cellscan be promoted, while the promotion of HEF cells autophagy can promote theproliferation of Toxoplasma gondii in host cells, therefore, the regulation on autophagyof host cell could regulate the proliferation and replication of Toxoplasma gondii.