RESUMO
Objective:To develop and optimize a novel assay for determination of cytotoxicity based Calcein -AM release.Methods:The target cells stained by Calcein-AM dye,then effectors and targets were incubated at E/T ratios from 30∶1-1∶1 for 4 h at 37℃,and the supernatant of reactions were detected by Fluorescence-Measurement to analyze specific cytotoxity.Results:The optimal excitation and emission wave lengths of Calcein were 485 nm and 515 nm.Dilutions of target cells stained by Calcein-AM had a linear relationship with measured fluorescence values.The Calcein-AM dye used to stain the living cells was shown to have a low spontaneous leakage rate-less than 15% in 4 hours at 37℃.Cytotoxicity activity of CIK showed a significant and positive correlation with E/T ratio when incubated at 4 h.Conclusion:The developed cytotoxicity test by Calcein-AM release is accurate and can avoid the application of radioactive reagents.
RESUMO
p53, as a transcription factor, is an important tumor suppressor gene and plays the key role in the p53-dependent gene regulatory network. Therefore, it is important to understand its biological function at the level of the whole system. In this paper, based on KEGG database and related literatures in English and Chinese, the interaction mode and quantitative relationship of the related molecules involved in p53 signaling pathway were extracted. By using S-system equations and 'Simulink' toolbox of Matlab7.0, a dynamic model of p53 signaling pathway was developed, and the dynamic regulatory characteristics of p53 signaling pathway were analyzed on model simulation. The results were in accord with the literatures and could reflect quantitatively the complex regulatory relationship between the interacting molecules involved in p53 signaling pathway. In addition, model simulation helped us find and identify the key molecules in this signaling pathway. Thus, this model can be used as a basis for the follow-up study of the relationship by precise and quantitative assessment.