孕期低剂量镉暴露对胎儿肝脏、胰腺和脂肪组织细胞凋亡功能的影响机制

The intergenerational exposure of cadmium has many toxicological effects on offsprings, and cadmium is one of the most toxic environmental pollutants that cause the developmental restriction of fetal tissues. However, the molecular mechanisms underlying cadmium toxicity to fetal development remain poorly understood. Our previous work has showed that low dose of gestational cadmium exposure can reduce the weight of fetal liver, pancreas and adipose tissues. Cell apoptosis has been identified as a vital regulator of tissue weight, and is closely associated with the developmental restriction of fetal tissues. In this study, TUNEL immunostaining of fetal liver, pancreas and adipose tissues will be performed to investigate whether gestational cadmium exposure can cause apoptotic death of fetal cells. The methods of the next generation sequencing, quantitative reverse transcription-polymerase chain reaction and Western blotting will be used to scan the differentially expressed genes/proteins in fetal tissues exposed to cadmium. In situ hybridization and bisulfate-sequencing PCR will be performed to reveal the localizations and the epigenetic regulation mechanisms of these differentially-expressed genes. Further in vivo functional studies are also planned to figure out the roles of these differentially-expressed genes in the cell apoptosis of fetal liver, pancreas and adipose tissues exposed to cadmium. We aim to show the molecular mechanisms of fetal tissue developmental restriction induced by gestational cadmium exposure, and provide a basis for the health risk assessment and the environmental management of cadmium exposure.

镉跨代暴露可对子代造成多种毒性效应，也是诱发胎儿器官发育受限的重要环境因素，但诱发受限的功能机制尚不清楚。项目申请人前期研究发现孕期低剂量镉暴露可引起胎儿的肝脏、胰腺和脂肪组织重量显著降低。细胞凋亡功能是决定器官重量的关键因素，与孕期低剂量镉暴露诱发的胎儿器官发育受限相关紧密。本项目拟一方面通过对孕期低剂量经口镉暴露胎鼠的肝脏等器官进行TUNEL免疫染色，探明孕期母体镉暴露对胎儿肝脏等器官的细胞凋亡功能的影响。另一方面通过二代测序等手段筛选差异表达基因。并通过免疫组化技术和启动子区DNA甲基化水平的分析，确定差异表达基因的定位模式和表观遗传学调控机制；同时，通过体内实验验证差异表达基因在孕期低剂量镉暴露介导的胎儿肝脏等组织的细胞凋亡功能中的调控作用。该研究旨在阐明孕期低剂量镉暴露对胎儿器官发育的毒性效应机制，为镉暴露健康风险评估和环境管理提供理论依据。

为探究镉对胎儿肝脏发育的毒性作用.拟选用15只性成熟的8周龄的雌性C57BL/6小鼠，与雄鼠交配见栓后，于妊娠第7.5 d(Embryonic 7.5，E7.5)时，随机分为 3 组(每组 5 只)，分别通过饮用水暴露法饲喂 0、20 和 40 mg·L-1 的氯化镉溶液，并于 E14.5 d 时收集各组小鼠的胎儿肝 脏等组织.通过 Ki67 和 TUNEL 染色方法探究镉对不同性别胎鼠肝脏的增殖功能和凋亡功能的影响，并通过 PCR 芯片筛查和实时定量 PCR 方 法检测镉处理组的雌性和雄性胎鼠肝脏中与细胞增殖和凋亡功能相关基因的表达变化.此外，我们还将通过石墨炉火焰原子吸收技术对镉处 理组的雌性和雄性胎鼠的镉含量进行检测.结果显示:孕期低剂量镉暴露对胎儿体重、胎肝重量和肝脏指数均无显著性影响.在本实验条件下， 镉对胎鼠肝脏的凋亡功能无显著性影响，但对增殖功能具有抑制作用，且该抑制作用具有雌性依赖性的特征.镉特异性诱导雌性胎鼠肝脏中 NF1 等多个细胞增殖功能相关基因以及 TGF⁃β1 信号通路分子的表达失衡;但对雄性胎鼠肝脏中相关基因的表达水平无显著性影响.镉在雌性胎 儿组织中的积累量显著高于雄性胎儿组织.综上所述，孕期低剂量镉暴露特异性抑制雌性胎儿肝脏的细胞增殖功能，并诱导雌性胎儿肝脏中 NF1 等多个增殖功能相关基因以及 TGF⁃β1 信号通路分子的表达失衡，该性别依赖性的抑制作用可能与镉在雌性胎儿中相对较高的积累量相关.