利用改良胚胎干细胞试验测定双酚A和金属镉的胚胎发育毒性-公共卫生与预防医学专业论文.docxVIP

ABSTRACTObjective：The ABSTRACT Objective：The embryonic stem cell test(EST)，a validated method，is used to assess the embryotoxic hazard of test substances in vitro．However,as the misclassifications of methylmercury,the generic limitation of EST became clear to US．This study was aimed to establish modified embryonic stem cell test system using the neuronal differentiation end point to substitute myocardial differentiation end point，and to apply the established experimental system to determine the developmental toxicity of bisphenol A and cadmium chloride． Methods：①The establishment and validation of neural differentiation method：The mRNA expression level of neural cell specific markers MAP2，nestin，and KCC2 during neural differentiation were detected by fluorescence quantitative PCR to explore the best embryonic stem cells inoculum density and culture time in nerve cell culture fluid；the protein expression level of neural cell specific markers MAP2，nestin，KCC2 and 13-Tubullin 111 were detected by immunofluorescence to determine the feasibility of neural differentiation method．②The establishment of modified EST：The established neural differentiation method was combined with the classical EST and was applied to determine the embryonic developmental toxicity of bisphenol A and cadmium chloride．In order to detect the credibility of the system，the positive control 5-Fu and negative control Penicillin-G were set up quality control group． Results：QThe most suitable embryonic stem cells inoculum density was 15 X 104／ well and the culture time in nerve cell culture fluid Was determined as 1 2 days． Compared to MAP2 and KCC2，Nestin was more suitable the detection biomarker of neural differentiation inhibition test．The results of immunofluorescence showed that the nerve cell specific markers were almost expressed in all cells，which showed that the neural differentiation method was feasible．②The modified EST with neural differentiation terminal was used to determine the deve