摘要
目的通过小鼠胚胎干细胞骨分化实验研究Cry1Ab蛋白对胚胎干细胞成骨分化过程的影响,以评估Cry1Ab蛋白的发育毒性。方法以Cry1Ab蛋白为受试物,设置5个剂量(125,250,320,1000,2000 ng/ml)染毒小鼠胚胎干细胞形成的拟胚胎体(embryonic body,EB),设置溶剂对照和阳性对照,同时向培养基中添加β-甘油磷酸盐(10 mmol/L)、抗坏血酸(50μg/ml)和维生素D3(500μmol/L)诱导EB向骨细胞分化,通过茜素红S染色和吸光度检测确定骨细胞钙化结节生成情况;收集EB制备细胞裂解液,通过BCA法测量细胞总蛋白浓度,采用碱性磷酸酶(alkaline phosphatase,ALP)检测试剂盒检测细胞裂解液中ALP活性;收集EB样本提取总RNA,通过qPCR检测骨细胞分化相关标志物Runx2、SPARC和I型胶原的基因表达情况。结果与对照组相比,不同浓度Cry1Ab蛋白处理组的EB细胞团大小、钙化结节数量无统计学差异(P>0.05);Cry1Ab蛋白各浓度组的细胞总蛋白浓度和ALP活性与对照组相比无统计学差异(P>0.05);Cry1Ab蛋白各浓度组的Runx2、SPARC和I型胶原基因表达水平与对照组相比均无统计学差异(P>0.05)。结论本次试验中,未观察到Cry1Ab蛋白对小鼠胚胎干细胞成骨分化过程产生影响,125~2000 ng/ml的Cry1Ab蛋白不具有发育毒性。
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(50μg/ml),and vitamin D3(500μmol/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.
作者
简远志
王菲
周若宇
尹宁
王军波
JIAN Yuan-zhi;WANG Fei;ZHOU Ruo-yu;YIN Ning;WANG Jun-bo(School of Public Health,Peking University,Beijing 100191,China;Beijing Key Laboratory of Toxicological Research and Risk Assessment for Food Safety,Beijing 100191,China)
出处
《食品与药品》
CAS
2024年第2期97-102,共6页
Food and Drug
基金
农业部转基因生物新品种培育重大专项(编号:2016ZX08011005)。
关键词
Cry1Ab蛋白
成骨分化
发育毒性
胚胎干细胞
Cry1Ab protein
osteogenic differentiation
developmental toxicity
embryonic stem cell
作者简介
简远志,硕士研究生,主要研究方向为食品安全,E-mail:1529436877@qq.com;通讯作者:王军波,副教授,医学博士,主要研究方向为营养与慢性疾病、婴幼儿营养、功能食品、食品安全,E-mail:bmuwjbxy@bjmu.edu.cn。
