镉对人胚胎神经干细胞增殖抑制及对金属硫蛋白各亚型的诱导表达  

Inhibition of Cell Proliferation and Expression of Metallothionein Gene Isoforms Induced by Cadmium in Human Embryonic Neural Stem Cells

作　　者：王新金[1] 窦婷婷[1] 吕文[1] 常秀丽[1] 周志俊[1] 

机构地区：[1]复旦大学公共卫生学院公共卫生安全国家重点实验室,上海200032

出　　处：《环境与职业医学》2015年第10期921-925,共5页Journal of Environmental and Occupational Medicine

基　　金：国家自然科学基金项目(编号:81472996);上海自然科学基金项目(编号:10ZR1401700)

摘　　要：[目的]探讨镉对人胚胎神经干细胞增殖能力的影响和对金属硫蛋白(MT)各亚型的诱导表达。[方法]以0、1.25、2.5、5μmol/L氯化镉(Cd Cl2)染毒人胚胎神经干细胞24 h后,以四甲基偶氮唑盐(MTT)法测定细胞活力,5-乙炔基-2′脱氧尿苷(Ed U)法测定细胞增殖能力,分光光度法测定培养上清中乳酸脱氢酶活性(LDH),应用逆转录多聚酶链反应(RT-PCR)检测镉对人胚胎神经干细胞MT 10个亚型的m RNA表达的影响。[结果]与对照组相比,浓度为2.5、5μmol/L Cd Cl2染毒组细胞活力明显降低,差异具有统计学意义(P<0.01);且细胞相对存活率与Cd Cl2浓度呈负相关(r=-0.98,P<0.01)。1.25μmol/L及以上镉可明显抑制DNA的复制(P<0.05),且细胞增殖与Cd Cl2浓度对数值呈负相关(r=-0.91,P<0.01)。与对照组相比,各染毒组细胞培养上清中LDH水平均高于对照组,差异有统计学意义(P<0.01),且细胞培养上清中LDH水平与Cd Cl2浓度呈正相关(r=0.97,P<0.01)。与对照组相比,MT基因各亚型的m RNA表达水平随着Cd Cl2染毒浓度的升高而升高,差异均有统计学意义(均P<0.05)。[结论]镉可引起人神经胚胎干细胞自我更新功能的抑制,表现为细胞活力和增殖能力的下降,细胞培养液中LDH含量的上升,细胞MT基因各亚型m RNA表达水平的明显升高。[ Objective ] To examine the effects of cadmium on self-renewal ability and expression of metallothionein （MT） gene isoforms of human embryonic neural stem cells （hNSCs）. [ Methods ] Treatment with 0, 1.25, 2.5, and 5 μmol/L cadmium chloride （CdCl2） for 24h was provided for hNSCs. Cell viability was measured by methyl thiazolyl tetrazolium （MTT） assay, cell proliferation by 5-ethynyl-2＇-deoxyuridine（EdU） assay, and lactic acid dehydrogenase （LDH） activity by spectrophotometric method. The mRNA expression of 10 MT isoforms in hNSCs after exposure to cadmium was measured by quantitative reverse transcription polymerase chain reaction （RT-PCR）. [ Results ] Compared with the control group, treatment with 2.5 and 5 μmol/L CdC12 for 24h significantly reduced the cell viability （P〈0.01）; the cell viability was negatively correlated with the concentrations of CdC12 （r=-0.98, P〈 0.01）. Treatment with 1.25 ~tmol/L and above remarkably inhibited DNA replication （P〈 0.05）, and the cell proliferation was negatively correlated with log-transformed CdC12 concentrations （r=-0.91, P〈 0.01）. Compared with the control group, all CdCl2 treatments for 24 h significantly increased the activities of LDH in supernatant （all P 〈 0.01）, and the activities of LDH and the concentration of CdCl2 showed a positive correlation （r=0.97, P 〈 0.01）. The mRNA expression levels of MT gene isoforms were significantly increased with higher concentrations of CdCl2 （P 〈 0.05）. [ Conclusion ] Cadmium could restrain the self-renewal ability of hNSCs via cell viability reduction, LDH activity elevation in hNSCs culture medium, and significant up-regulated mRNA expression of MT gene isoforms.

关 键 词：镉 人胚胎神经干细胞 细胞增殖 金属硫蛋白 神经发育毒性 

分 类 号：R114[医药卫生—卫生毒理学]