论文部分内容阅读
目的探究孕期镉暴露对小鼠胎盘氨基酸转运系统的影响及其作用机制。方法选择45只体重相近的孕鼠,于妊娠第7.5天(Embryonic 7.5,E7.5)时,随机(完全随机化法)分为3组,分别喂食0,20mg/L和40mg/L的氯化镉溶液。分别于E13.5天、E16.5天和E19.5天收集各组小鼠的胎盘组织标本和血浆样本,每一妊娠阶段5只动物。通过实时定量PCR技术检测各组小鼠胎盘中L型转运载体-1(L-type amino acid transporter 1,LAT1)、L型转运载体-2(L-type amino acid transporter 2,LAT2)、L型转运载体-3(L-type amino acid transporter 3,LAT3)、L型转运载体-4(L-type amino acid transporter 4,LAT4)、A型转运载体-1(sodium-coupled neutral amino acid transporter 1,SNAT1)和A型转运载体-2(sodium-coupled neutral amino acid transporter 2,SNAT2)的m RNA表达情况,并对其中差异表达的分子进一步通过蛋白印迹法鉴定其蛋白质水平的变化情况。此外,采用可见光比色法检测各组小鼠血浆中氨基酸的含量。结果 (1)3组孕鼠胎盘中LAT1、LAT2、LAT3、LAT4、SNAT1和SNAT2的m RNA表达水平的比较:3组孕鼠胎盘中的LAT1分子,E19.5天时在40mg/L镉处理组的小鼠胎盘中的m RNA表达水平与正常对照组相比显著性降低,而在E16.5天时无显著性变化。其他5种氨基酸转运载体在不同浓度镉处理组的小鼠胎盘中的m RNA表达水平与正常对照组相比,在E16.5天和E19.5天时均没有显著性变化。(2)3组孕鼠胎盘中LAT1蛋白质表达水平的比较:E16.5天时,与正常对照组相比,不同浓度镉处理组小鼠胎盘中的LAT1蛋白表达水平出现降低,并且40mg/L镉处理组的小鼠胎盘中的LAT1蛋白质表达水平出现显著性变化。E19.5天时,与正常对照组相比,20mg/L和40mg/L镉处理组小鼠胎盘的LAT1蛋白表达水平均明显降低。(3)3组孕鼠血浆中氨基酸含量的比较:E19.5天时,不同浓度镉处理组的小鼠血浆中氨基酸含量与正常对照组相比均出现降低,并且40mg/L镉处理组的小鼠血浆中的氨基酸含量发生显著性变化。E16.5天时,不同浓度镉处理组的小鼠血浆的氨基酸含量与正常对照组相比,均无显著性变化。结论孕期镉暴露引起小鼠胎盘中氨基酸转运载体LAT1的m RNA和蛋白质表达水平和血浆氨基酸含量的显著性降低,这可能是镉致胎儿生长受限的一个重要因素。此外,血浆中氨基酸浓度的降低可能对胎盘中氨基酸转运载体的表达产生抑制作用。
Objective To investigate the effect of gestational cadmium exposure on the placental amino acid transport system in mice and its mechanism. Methods Forty-five pregnant rats of similar body weight were randomly divided into three groups on the 7.5th day of gestation (Embryonic 7.5, E7.5), fed with 0, 20mg / L and 40mg / L of chlorine respectively Cadmium solution. Placental tissue samples and plasma samples from each group of mice were collected on days E13.5, E16.5 and E19.5, respectively, and five animals per gestation stage. Real-time quantitative PCR was used to detect the expression of L-type amino acid transporter 1 (LAT1), L-type amino acid transporter 2 (LAT2), L-type L-type amino acid transporter 3 (LAT3), L-type amino acid transporter 4 (LAT4), sodium-coupled neutral amino acid transporter 1 SNAT1 and SNAT2 were detected by RT-PCR. The differentially expressed proteins were further identified by Western blotting. In addition, the contents of amino acids in the plasma of mice in each group were detected by visible light colorimetry. Results (1) Comparison of mRNA expression levels of LAT1, LAT2, LAT3, LAT4, SNAT1 and SNAT2 in placenta of three groups: LAT1 molecules in placenta of pregnant rats in three groups were treated with 40 mg / L Cd M RNA expression in the mouse placenta was significantly lower than that in the normal control group, but not significantly changed at E16.5 days. Compared with the normal control group, m RNA expression levels of other 5 kinds of amino acid transporters in mouse placenta treated with different concentrations of cadmium did not change significantly at E16.5 days and E19.5 days. (2) Comparison of LAT1protein expression in placenta of three groups: At E16.5days, compared with the normal control group, the expression level of LAT1protein in the placenta of mice treated with different concentrations of cadmium decreased, and the concentration of40mg / L cadmium There was a significant change in LAT1 protein expression in the treated group of mouse placenta. E19.5 days, compared with the normal control group, 20mg / L and 40mg / L cadmium treatment group of mouse placenta LAT1 protein expression levels were significantly lower. (3) Comparison of amino acid content in the plasma of three groups of pregnant rats: At E19.5, the amino acid content in the plasma of mice treated with different concentrations of cadmium decreased compared with the normal control group, and the small Amino acid content in rat plasma changed significantly. E16.5 days, different concentrations of cadmium in mice plasma amino acid content compared with the normal control group, no significant change. Conclusion Exposure to cadmium during pregnancy induces a significant decrease in m RNA and protein expression of the amino acid transporter LAT1 and plasma amino acid levels in the placenta of mice, which may be an important factor of fetal growth restriction caused by cadmium. In addition, a decrease in plasma amino acid concentration may have an inhibitory effect on the expression of the amino acid transporter in the placenta.