【摘要】 目前,药品安全、食品安全和环境污染等问题受到国人的广泛关注,也受到政府的特别重视。医用化学品、食品添加剂和环境污染物等通过各种途径进入体内,可能产生胚胎发育毒性;因此,建立新的高通量、高灵敏的胚胎发育毒性检测和评价十分重要。研究尝试利用全反式维甲酸(all-trans retinoic acid,RA)对人胚胎干细胞H9的细胞毒性和分化抑制,评价RA的胚胎发育毒性。通过CCK8检测不同浓度下RA对人胚胎干细胞H9和鼠胚胎成纤维细胞3T3的细胞存活百分数和半数增殖抑制浓度(50%inhibitory concentration,IC50),发现处理5 d和10 d,RA对人胚胎干细胞H9的IC50分别是9.23μg/mL和7.20μg/mL,明显低于3T3细胞。通过实时定量PCR检测不同RA浓度下,自然分化20 d的细胞中Nkx2.5、α-MHC、ACTC1和TNNT2基因的表达,发现0.3μg/mL及其以上浓度RA显著抑制H9细胞向心肌细胞分化。RA对H9细胞α-MHC、TNNT2和ACTC1的半数抑制分化浓度(ID50),分别为0.16、0.07和0.05μg/mL,ACTC1和TNNT2的ID50明显低于α-MHC的,提示ACTC1和TNNT2可能更适合作为人胚胎干细胞分化抑制的评价的指标。更多还原

【Abstract】 Recently,the safety of drug and food and environment pollution draws more and more attention.Medical products,food additives and pollutants could enter the body via various manners. Thus,it’s important to establish a new high throughout model for evaluating developmental toxicity. To investigate develop-mental toxicity of retinoic acid,the cytotoxicity and the potential inhibition of differentiation were studied. CCK8 was used to detect the 50% proliferation inhibitory concentration( IC50) of human embryonic stem cell-H9 and mouse embryo fibroblasts-3T3. The result showed that IC50H9-D5 and IC50H9-D10 is 9. 23 and 7. 20 μg /mL which were significantly higher than IC503T3-D5 and IC503T3-D10. Real-time PCR was used to detect the expression of Nkx2. 5,α-MHC,ACTC1 and TNNT2 after H9 cells were treated with RA. The results showed that RA significantly inhibited differentiation of H9 s to myocardial cells above the dosage of 0. 3 μg /mL( including 0. 3 μg /mL). ID50H9 of α-MHC,ACTC1 and TNNT2 was 0. 16,0. 07 and 0. 05 μg /mL. ID50H9 of ACTC1 and TNNT2 were similar,but they were lower than ID50H9 of α-MHC. These suggested that ACTC1 and TNNT2 could be good monitoring markers for detecting the inhibition of differentiation.更多还原