Preliminary Study For Chicken Prion On The Regulation Of DF-1Cell Activity

【Objective】Through over-expression of chick prion protein (ChPrP) in DF-1cell, this researchpreliminarily explorated ChPrP on the influence of proliferation, adherency, migration and apoptosis in DF-1cell, and detected the expression level of Akt related to proliferation betwwen DF-1cell with over-expressionof ChPrP and normal DF-1cell. Adding the Wortmannin for blocking the PI3K/Akt signal pathway, this studyshowed the molecular mechanism that over-expression of chick prion regulated DF-1cells, and moreknowing the biofunction of chicken prion.This proposal will provide a new theoretical basis for the study oftumour in chicken.【Methods】(1)According to sequences of β-actin and Akt in GenBank, two pairs of primers weredesigned,respectively.After optimizing the conditions, a reverse transcription fluorescence quantified PCRwas established for Akt mRNA. After extracting the total RNA from DF-1, DF-1-cont and DF-1-PrP, AktmRNA was detected by this method, and then data was analyzed by spss.(2) After adding the Wortmannininto DF-1-PrP、DF-1-cont and DF-1, proliferation, adherency, migration and apoptosis was detected by MTT,and transwell for the influence of prion,respectively.The expression level of prion was detected by PRNPreverse transcription fluorescence quantified PCR in different levels of Wortmannin.【Results】(1) The double standard curve method of Akt and β-actin was established based on the SYBRGreen I real-time fluorescence quantitative PCR. Standard curves of pMD18-Akt and pMD18-β-actin werey=-3.473x+42.78, y=-3.506x+44.18, respectively, and the sensitivity of them were104. The samples weredetected by our established RT-PCR method, displaying stably and reliably and good specificity with specificand single amplification products. Using this method to separately detect the Akt transcription levels in DF-1-PrP cells and DF-1-count cells, the former is1.6569x103and the latter is2.2574x102. The Akt expressionlevels of DF-1-PrP cell was obviously higher than that of DF-1-count cell (p <0.01), implying that theexpressing quantity of PrPC are positively correlated with Akt in DF-1cell. This method was used to detectthe Akt gene transcription level for accessing the proliferation of DF-1cells (2) The cell proliferation,adhesion, and invasion assay showed that the cell proliferation, adhesion and invasion assay of allexperimental groups were lower than the control. As the wortmannin concentration was no more than20nmol/L, effects were little on proliferation, adhesion and invasion; while the concentration was no less than50nmoL/L, the proliferation, adhesion and invasion were obviously inhibited. However, when thewortmannin concentration was no more than50nmol/L, DF-1-PrP cells were less inhibited by wortmannindue to over-expressed PrPC.Additonally,, the copy numbers of PrPC gene mRNA in three kinds of DF-1celltreating with wortmannin showed that the expression levels of PrPC in three kinds of DF-1cell were varied,and PrPC expression levels generally presented a downward trend with the addition of wortmannin.【Conclusions】(1) The expression level of Akt gene is closely related to PRNP,indicating that ChPrPCinvolved in the activation of Akt, and Akt probably exist feedback regulation on PrPC.(2) ChPrPCinvolved in the proliferation, adhesion, invasion and apoptosis of DF-1cells, which could promote DF-1cell proliferation,inhibit DF-1cell apoptosis and improve the adhesion and invasion of DF-1cell.(3) The wortmannin, aninhibitor on PI3K/Akt signal pathway, obviously inhibit the proliferation, adhesion, invasion of DF-1cell,and promote cell apoptosis, and the effect was positively correlated to drug concentrations. Compared toDF-1cells, DF-1-PrP cells were less influenced by wortmannin (50nmol/L), indicating that the proliferation,adhesion, invasion of DF-1cell are regulated by over-expressed PrPCin some other ways.