Toxoplasma Gondii Inhibits The Differentiation Of C17.2 Neural Stem Cells Through Wnt/β-catenin Pathway

Objective To explore the effects of Toxoplasma gondii excretory-secretory antigens(ESAs) on the differentiation of neural stem cell and its mechanism. Methods(1) To optimize the differentiation of C17.2 neural stem cells, the cells were cultured using three different culture medium, including DMEM complete medium sup-plemented with 5% horse serum and 10% fetal bovine serum, serum-free DMEM cultu- re medium, and serum-free DMEM/F12 medium with 2% N2 supplements respectively. Observing morphology changes of C17.2 cells every day, and detecting the m RNA levels of βⅢ-tubulin using RT-PCR on day 1, day 3 and day 5(2) T. gondii ESAs were added into C17.2 culture medium with DMEM/F12 plus 2% N2, and after 5 days co-culture the cells were harvested and the protein levels of βⅢ-tubulin and GFAP were identified using Immunofluorescence and Western blotting.(3) C17.2 cells were cultured with serum-free DMEM/F12 plus 2% N2, and then three different doses of T. gondii ESAs(0.07mg/ml,0.14mg/ml,0.28mg/ml) were added respectively. On the day 5 the cells were harvested and the protein levels of βⅢ-tubulin and GFAP were analyzed using western blotting.(4) C17.2 cells were pretreated with Wnt pathways activator Wnt3 a, and then cultured in DMEM/F12 plus 2% N2 with or without addition of 0.28mg/ml ESAs. Wnt pathways related protein β-catenin Ngn1 and Ngn2 were detected using Western blotting. Results(1) C17.2 neural stem cells cultured in DMEM / F12 with 2% N2 supplements obtained two different morphological cell types, assumed to be neurons and gial cells. And the m RNA level of βⅢ-tubulin in C17.2 cells when cultured in DMEM / F12 with 2% N2 supplements(3.93±0.13) was significantly higher than that when cultured in DMEM complete medium(0.08±0.34%) and serum-free DMEM culture medium(0)on day 5.(2) Moreover, when cultured in DMEM / F12 medium with 2% N2 for 5 days, the protein level of βⅢ-tubulin and GFAP in C17.2 neural stem cells was significantly down-regulated by adding the ESAs of T. gondii RH.(3)The protein level of βⅢ-tubulin in C17.2 neural stem cells cultured with three different doses of T. gondii ESAs(0.07mg/ml,0.14mg/ml,0.28mg/ml)for 5 days was 0.617±0.058,0.290±0.072,0.043±0.006,respectively. All were found significantly lower than thant in control group(1.507±0.092).Accordingly, the protein level of GFAP in C17.2 neural stem cells treated with three different doses of T. gondii ESAs was 0.069±0.06,0.040±0.003,0.029±0.004,respectively, also was found significantly lower than that in control group( 0.704±0.036). The data displayed that that ESAs of T. gondii RH inhibits the differentiation of C17.2 cells in a dose-dependent manner.(4) After C17.2 cells were pretreated with Wnt3 a, ESAs and Wnt3 a plus ESAs, The protein level of β-catenin in the nuclear was 0.033±0.006,0.140±0.026,0.081±0.003,respectively,and downstream target genes Neurogenin1 in the cells was 0.029±0.014,2.507±0.060,1.603±0.451,and downstream target genes Neurogenin2 in the cells was 0.041±0.006,0.117±0.011,0.069 ±0.035. The data showed the ESAs reversed the activation effect of Wnt3 a on C17.2 cells differention. Conclusion(1) The ESAs of RH T. gondii inhibits the differentiation of C17.2 neural stem cells.(2) Toxoplasma gongdii inhibits the differentiation of C17.2 neural stem cells may be through Wnt/β-catenin Pathway.