Establishment Of Mouse Embryonic Stem Cells Test Model Used For Screening Developmental Toxicity Of AFGF

[OBJECTIVE]To establish a model of mouse embryonic stem cell tests (EST) and verify the validity of the model detecting the embryonal toxicity. Besides, explore the embryonic developmental of acid fibroblast growth factor (aFGF) and investigate its influence on differentiation of mouse embryonic stem cell (ES cells).[METHODS]Mouse embryonic stem cells were cultured in vitro and identified by observing morphology, analyzing karyotype, staining alkaline phosphatase and so on. The cytotoxicity effect of 5-Fu, DPH and Penicillin G on ES and 3T3 cells were detected by MTT assay, and the effect of three drugs on undifferentiated Sox-2 gene expression were detected by semi-quantitative RT-PCR analysis to determine their inhibitory degree on ES cell differentiation, in accordance with the in vitro embryotoxicity evaluation method which was recommended by European Centre for the validation alternative test methods (ECVAM), and thus verify the effectiveness of the EST model on developmental toxicity of drugs as well as evaluate the embryotoxicity of aFGF(14-154). Finally, the expressions of specific molecular markers on the ES cell in differentiation of three germ layers were determined by RT-PCR with different doses of aFGF.[RESULTS]EST model was established successfully. As the same with the clinical toxicities of the three drugs, the embryotoxicity of 5-Fu were strong, that of DHP was weak and no embryotoxicity of Penicillin G was observed. Within the scope of 0.01-100μg/mL, aFGF (14-154) have a mitogenic effect on 3T3 cells and ES cells, but have various degrees of inhibitory effect on them with the concentration above 100μg/ml; at the concentration of 0.01-100μg/mL, aFGF inhibited the ES cell differentiation. The IC50 of aFGF (14-154) on 3T3 cells was 263.786μg/mL; while IC50 and ID50 on ES cells were 393.12μg/mL and 6.42μg/mL respectively, the results indicated that aFGF posses a weak embryotoxicity. At relative low doses of aFGF (14-154) can promote the expression of ectoderm markers:GFAP, Oligo2, Nestin, and reached the highest value at 1μg/mL of aFGF of all. With the concentration increased, its promoter action would decline; the tendencies are inconsistent on the typical mesoderm marker:BMP4, MHC, MyoD; while there are significantly inhibited on endoderm markers:GATA6, TTR and ALB.[CONCLUSION]The established EST model can be used for preliminary evaluating embryonal and developmental toxicity of drugs. The evaluated result showed that aFGF possessed a weak embryotoxicity. The effect of aFGF(14-154) on the development of the three germ layers showed concentration-dependent, and the roles of aFGF on the three germ layers are inconsistent or even contrary at the same dose, this imbalance may be related to the role of weak developmental toxicity The expression inhibition of endodermal markers:GATA6, TTR, and ALB gene in determining the embryotoxicity of aFGF may have significance.