| Neosporosis is a protozoosis caused by Neospora caninum, which has awordwide distribution.N.caninum can infect many kinds of animals, such as canine,cattle, goats, sheep, horses, deer, fox and so on. It can cause meningoencephalitis,polymyositis, polyradiculoneuritis and abortion, stillbirth, foetal mummification ofpregnant animals and movement dysfunction of newborns, which cause greateconomic losses to livestock and aquaculture.EGFR signaling pathway is an important signaling pathway in the life activity,which can regulate cell proliferation, apoptosis, migration, survival, and a series ofcomplex processes. Research found that toxoplasma gondii infection early canactivate the host cell EGFR signaling pathways, preventing autophagyprotein-mediated killing of the parasite. But N.caninum invasion’s effect on EGFRsignaling pathway of host cells has not been reported. This experiment make asystematic study of establishing the method of N.caninum tachyzoites in vitro culture,N.caninum invasion’ effect on the EGFR signaling pathway of the host cell,and theimpact of inhibiting the EGFR signal pathway on N.caninum invasion to illustratethe variations of host cells EGFR signaling pathway and the effect of EGFRsignaling pathway during the process of N.caninum invasion. This study hasimportant academic value and scientific significance to reveal the interactionmechanisms and pathogenesis mechanisms between N.caninum.This article mainly includes the following contents:The culture of N.caninum tachyzoites in vitro After N.caninum tachyzoiteswas cultured in the293T, HCT-8, Hela and Vero cells, We observed and counted thenumber of tachyzoites, and drawn the growth curve.We stained the tachyzoites withacridine orange and observed it by confocal microscope. The results showed that thetachyzoites grew faster in293T, HCT-8and Vero cells.Its number began to increase inthe third day of infection and peaked in the5,7and6days respectively. However, the tachyzoites grew slower in Hela cells and its number had been at a low level. Theparasitophorous vacuoles (PV) were larger and the number of tachyzoites in PV wasgreater in293T, HCT-8and Vero cells than those in Hela cells. The experimentprovided new cell lines (293T and HCT-8) for N.caninum tachyzoites culture in vitro.The effect of N.caninum invasion on the EGFR signaling pathway of thehost cell After inoculating N.caninum tachyzoites to293T cells, we collectedcells in different times (10min,20min,30min and60min), extracted the total RNAand protein, and used Real time PCR and Western Blot method to detect the changesof EGFR and its downstream PI3K/Akt, cell apoptosis and cytoskeleton regulationpathway. The results showed that the N.caninum invasion can obviously increase thegene expression of EGFR, PI3K, Akt, Bad, Bcl-2, MUC1, Caveolin and E-cadherin,decrease of Bax and P53, but not affect of paxillin and β-catenin. N.caninuminvasion can phosphorylated EGFR (Tyr1173), Akt (Ser473) and Bad (Ser112).EGFR inhibitors AG1478can decrease phosphorylation of EGFR and Akt, and PI3Kinhibitors LY294002can decrease the phosphorylation of Akt and Bad of host cellafter N.caninum invasion. In conclusion, the invasion of N.caninum tachyzoites canactivate the EGFR signaling pathway of host cell.The impact of inhibiting the EGFR signal pathway on N.caninum invasion293T cells treated with EGFR signal pathway inhibitor (EGFR inhibitors AG1478,PI3K inhibitors LY294002and Akt inhibitors IV) were infected with N.caninumtachyzoites, than we observed the number of infected cells and tachyzoites in theinfected cells. The results showed that after24h of infection, the number of infectedcells and the number of tachyzoites decreased significantly in cells treated withAG1478and IV, but cells treated with LY294002had no obvious change. Theresults showed that the activation of EGFR signal pathway of host cell was related toN.caninum invasion. |
