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
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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、5 μmol/L 氯化镉(CdCl2)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(EdU)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mRNA表达的影响。 结果 与对照组相比, 浓度为2.5、5 μmol/L CdCl2 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CdCl2 浓度呈负相关(r=-0.98, P < 0.01)。1.25 μmol/L及以上镉可明显抑制DNA的复制(P < 0.05), 且细胞增殖与CdCl2 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CdCl2 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mRNA表达水平随着CdCl2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mRNA表达水平的明显升高。 Abstract: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 24 h 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 CdCl2 for 24 h significantly reduced the cell viability (P < 0.01); the cell viability was negatively correlated with the concentrations of CdCl2 (r=-0.98, P < 0.01). Treatment with 1.25 μmol/L and above remarkably inhibited DNA replication (P < 0.05), and the cell proliferation was negatively correlated with log-transformed CdCl2 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.
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