Evaluation of Developmental Toxicity of Cry1Ab Protein Through Embryonic Stem Cell Osteogenic Differentiation Experiments
Objective To investigate the effect of Cry1Ab protein on the osteogenic differentiation process of mouse embryonic stem cells in vitro to assess its developmental toxicity.Methods Cry1Ab protein was used as the test substance and 5 doses(125,250,320,1000,2000 ng/ml)of Cry1Ab protein were administered to embryonic bodies(EBs)formed by embryonic stem cells.Solvent control(SC)and positive control(PC)groups were also set.β-glycerophosphate(10 mmol/L),ascorbic acid(50 μg/ml),and vitamin D3(500 μmol/L)were added to the culture medium to induce osteoblast differentiation.The formation of osteoblast calcified nodules was determined by alizarin red S staining and absorbance detection.Samples of EBs were collected to prepare cell lysates,and the total protein concentration was measured by the BCA method.The activity of alkaline phosphatase(ALP)in the cell lysate was detected by an ALP test kit.Total RNA was extracted from EBs samples,and qPCR was used to detect the expression of osteoblast differentiation-related markers(Runx2,SPARC,type Ⅰ collagen).Results There were no statistically significant differences(P>0.05)between the size of EBs cell clusters and the number of calcified nodules of each Cry1Ab protein treated group and those of the SC group.Additionally,the differences between the total cellular protein concentration and ALP activity of each Cry1Ab protein-treated group and those of the SC group were not statistically significant(P>0.05).The gene expression levels of Runx2,SPARC,and type Ⅰ collagen in the Cry1Ab protein-treated groups at various concentrations were not significantly different(P>0.05)from those in the SC group.Conclusion In this experiment,no effects of Cry1Ab protein on osteogenic differentiation process of mouse embryonic stem cells are observed,and Cry1Ab protein at concentrations of 125-2000 ng/ml does not exhibit developmental toxicity.
万方数据
维普
