A Mechanistic Study Of Toxoplasma Rhoptry Kinase 18 On The Apoptosis And Differentiation Of Neural Stem Cell

Objective To explore the effects of Toxoplasma rhoptry kinase 18 on the apoptosis and differentiation of neural stem cell and its mechanism.Methods Toxoplasma gondii ROP18 was subcloned from PEGFP-G2-ROP18 into pHBAd-MCMV-GFP. The recombinant adenovirus vector pHBAd-MCMV-GFP-ROP18 was comfirmed by PCR and sequencing and then the identified recombinant adenovirus vector was transfected into 293 cells for packaging with skeleton plasmid. After amplification, ROP18 recombinant adenovirus (MOI=70) was transfected into C17.2 neural stem cells. The proliferation of C17.2 cells at 48h post treatment was detected using CCK-8 kit and the apoptosis of C 17.2 cells at 24h post treatment was detected by flow cytometer. Western blot was used to determine the expression levels of caspase-3. On day3 sand day5 after co-culture, the cells were harvested and the protein level of βⅢ-tubulin was identified using immunofluorescence and western blot. Wnt pathways related protein β-catenin, Ngnl and Ngn2 were detected by Western blot. The TOP/FOP promoter activity in C17.2 NSCs cells by luciferase.Results (1)The recombinant adenovirus vector pHBAd-MCMV-GFP-Ropl8 was successfully constructed and highly expressed in C17.2 neural stem cells.(2) Compared with the control group, proliferation of C17.2 cells was found to be significantly decreased (P<0.05) in the ROP18 recombinant adenovirus group at 48h post treatment, while the apoptosis of C17.2 cells was significantly increased (P<0.05) at 24h post treatment, and the expression level of caspase-3 was significantly increased(P<0.05) in the ROP18 recombinant adenovirus group at 24h post treatment. (3) When cultured in DMEM/F12 medium supplemented with 2% N2 for 3 days or 5 days, the levels of βⅢ-tubulin was significantly down-regulated by adding the ROP18 recombinant adenovirus.(4)The protein level of βⅢ-tubulin was significantly decreased (P<0.05) in the ROP18 recombinant adenovirus on day3 and day5 post treatment. (5)The protein levels of β-catenin both in nucleus and cytoplasma were significantly decreased (P<0.05) in C17.2 neural stem cells transfected by ROP18 recombinant adenovirus on day3 and day5 post treatment.Moreover, the protein levels of Ngnland Ngn2 were also found significantly decreased (P<0.05) in C17.2 neural stem cells transfected by ROP18 recombinant adenovirus on day3and day 5 post treatment. The expression of ROP18 was downregulated the TOP/FOP promoter activity in C 17.2 NSCs cells.Conclusion(1) ROP18 recombinant adenovirus was successfully constructed and highly expressed in C17.2 neural stem cells.(2) ROP18 recombinant adenovirus induced the apoptosis of C17.2 neural stem cells.(3) ROP18 recombinant adenovirus inhibited the differentiation of C 17.2 neural stem cells.(4) ROP 18 recombinant adenovirus may inhibit the differentiation of C17.2 neural stem cells through Wnt/β-catenin Pathway.