| 【Objective】 In this study, we used the established chicken prion protein verexpression,suppressed and normal expression of DF-1 cell lining in cell adhesion, invasion, proliferation and apoptosis, the initial explore the impact of avian prion protein on DF-1 cells; by detecting prion protein expression and suppressed expression of the prion protein related protein expression of CK2 and DF-1 cells and normal cells in DF-1 anti-apoptotic Bcl-2, and using CK2 mitoxantrone inhibitors block apoptosis-related signal transduction pathways(Wnt/β-catenin pathway), to clarify the prion protein overexpression and inhibition of expression of the molecular mechanisms of DF-1 cells, to learn more about birds of prion physiological function, for the study of poultry cell tumorigenesis mechanism to provide theoretical basis.【Methods】(1) According to Gen Bank of chicken CK2, Bcl-2 and β-actin gene sequence of each design a pair of primers, Optimized reaction conditions, the establishment of quantitative reverse transcription PCR m RNA Ch CK2 and Ch Bcl-2; will pass after generation stable collection DF-1-Pr P, DF-1 cell extracts and DPs3-5 total RNA, c DNA obtained by reverse transcription quantitative PCR methods described above was amplified, detected three kinds of cells CK2 and Bcl-2 gene expression differences.(2) in DF-1-Pr P, DF-1 and DPs3-5 cells with different concentrations of mitoxantrone, after a period of action, by cell MTT assay, flow cytometry, adhesion assay and invasion experiments Methods to detect prion protein CK2 on DF-1 cell proliferation, adhesion, invasion and apoptosis, and using laboratory established PRNP SYBR GreenⅠ double standard curve quantitative PCR method detects the expression of prion protein concentration of three kinds of cells in different hydrochloride meters under the anthraquinone concentration.【Results】(1) The establishment of a CK2, Bcl-2 and β-actin of SYBR Green I fluorescence quantitative RT-PCR assay double standard curve, won the best reaction system and conditions, has been the standard quality grain p MD18-CK2, p MD18-Bcl-2 and the standard curve p MD18-β-actin were y =-3.219 x + 46.2, y =-3.17 x + 45.52 and y =-3.24 + 43.62, sensitivity was 104. Established Ch CK2 RT-PCR standard curves single melting peak, a good linear relationship can be used for reliable detection of the sample. Use this method to detect the relative expression of DF-1-Pr P and DPs3-5 cell lines amount CK2 genes were 6.46 × 105 and 8.32 × 103, and the rative expression levels of Bcl-2 are 7.91×105 and 1.51×103, which shows the expression of CK2 and Bcl- 2 gene in DF-1-Pr P cell were significantly higher than DF-1 cells(p <0.05), indicating that the expression of CK2 and Bcl-2 in different DF-1 cells are positively correlated with the expression of Pr PC;(2) The experimental results of cell adhesion, proliferation and invasion showed that: the activity of the experimental group in cell adhesion, proliferation and invasion were lower than the control group, the activity of cells; when mitoxantrone final concentration of less than 25 n M, it had little effect for cells of adhesion and inhibition, and when it rises to a final concentration of 50 nmol / L, cell adhesion, the inhibition activity of proliferation and invasion increased significantly(P <0.05), and with the increasing of the concentration of mitoxantrone, this inhibition gradually increased, and showed dose-effect relationship. Apoptosis results show:the prion protein overexpression apoptosis rate DF-1-Pr P is lower than the normal prion protein DF-1 and inhibit the expression of DPs3-5, with mitoxantrone role 25,50 n M in DF-1, the rate of apoptosis gradually increased, while mitoxantrone concentrations below 100 n M, the promotion of apoptosis rate of cells of the prion protein over-expression was significantly smaller than the other two types of cells. Simultaneously, due to over-expression of Pr PC, DF-1-Pr P cells mitoxantrone concentration is less than 50 n M, which shows certain resistance effect, so that the DF-1-Pr P cells acquire resistance to mitoxantrone activity inhibitory capacity; in mitoxantrone effect, three kinds of DF-1 cells Pr PC gene m RNA copy number detection display, three kinds of DF-1 cells express Pr PC volume changes, with the concentrations of mitoxantrone increasing, expression of Pr PC generally showing a downward trend.【Conclusions】(1) The expression of Bcl-2 and CK2 gene is closely related to the amount of PRNP, suggesting that Ch Pr PC may be involved in the activation of CK2 and Bcl-2, and synergy with Pr PC;(2) Ch Pr PC involved in the process of DF-1 cell proliferation, adhesion, invasion and apoptosis, which can promote DF-1 cells of the proliferation,adhesion and invasion, and then inhibition the DF-1 cells of apoptosis;(3) adding Wnt signal transduction pathway of β-catenin After phosphorylation of CK2 inhibitor conjugate mitoxantrone, can significantly inhibit the proliferation the DF-1 cell of adhesion and invasion, and to promote the DF-1 cell apoptosis, and the effect was positively correlated with the concentration of the drug. Compared to normal cells of DF-1, DF-1-Pr P cells are mitoxantrone(concentration less than 50 n M) of the impact is small, which shows that the over-expression of Pr PC may regulate the proliferation, adhesion and invasion ability of DF-1 cells by other means. |
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