p53和MAPK信号通路在镉诱导猪胎盘滋养层细胞凋亡中的作用研究

Cadmium, a toxic heavy metal element which is easy to accumulate in placentae, can cause dysfunction of placentae and trophoblast cells，resulting in subsequent fetal growth retardation and even abortion, leading to serious negative effects on livestock reproduction and husbandry development. In recent years, porcine abortion caused by cadmium poisoning has been frequently reported. However, to present, the underlying cellular and molecular mechanisms regarding abortion caused by placental injury are not clear yet. In this study, pregnant sows and trophoblast cells cultured in vitro will be treated by cadmium. Pathological and sub microstructure change of placental trophoblast cells will be assessed both in vivo and in vitro by virtue of pathological and transmission electron microscopic observations. The generation of ROS, DNA damage and apoptosis will be analyzed by flow cytometry analysis and immunofluorescence. The involvement of p53 and MAPK signaling pathways under cadmium exposure will be investigated by Real-time PCR and Western blot. These results will reveal the cellular and molecular mechanisms of cadmium caused dysfunction of placentae. In our previous study, we have observed that cadmium can induce apoptosis of porcine trophoblast cells in vitro. We expect that the results obtained from this study will reveal the mechanisms of placental injury caused by cadmium, and provide theoretical support to solve the cadmium poisoning which threatens livestock reproduction and husbandry development.

镉是一种极易在胎盘内蓄积的有毒重金属元素，能够引起胎盘和胎盘滋养层细胞功能障碍，最终导致胎儿发育迟缓甚至流产，严重影响了家畜繁殖和畜牧业发展。近年来，镉中毒引起的妊娠母猪流产事件屡见报道，一般认为，这一流产事件是由于镉引起母猪胎盘损伤所导致。本项目组已经证实镉能够诱导体外培养的猪胎盘滋养层细胞发生凋亡，但具体的细胞分子机制尚不明确。本项目拟对妊娠母猪及体外培养的胎盘滋养层细胞进行镉胁迫，通过病理学检测和透射电镜观察，在动物和细胞水平研究胎盘滋养层细胞的病理变化和亚显微结构变化；采用流式细胞术和免疫荧光染色检测胎盘滋养层细胞中ROS生成、DNA损伤和细胞凋亡情况；Real-time PCR和Western blot检测p53和MAPK信号通路在胎盘滋养层细胞应答镉胁迫时的活化情况，最终阐明镉引起妊娠母猪胎盘损伤的细胞分子机制。研究结果将为防治危害家畜繁殖和畜牧业发展的镉中毒问题提供理论指导。

镉（Cadmium, Cd）是一种极易在胎盘内蓄积的有毒重金属元素，能够引起胎盘和胎盘滋养层细胞功能障碍，最终导致胎儿发育迟缓甚至流产，严重影响了家畜繁殖和畜牧业发展。近年来，Cd中毒引起的妊娠母猪流产事件屡见报道，一般认为，这一流产事件是由于Cd引起母猪胎盘损伤所导致。因此，本项目重点研究Cd对妊娠期母猪胎盘组织和胎盘滋养层细胞的毒性损伤，并对其分子机制进行深入探讨。首先，通过胶原酶消化法获得了原代猪胎盘滋养层细胞，以体外培养的原代细胞为实验模型，对Cd引起的猪胎盘滋养层细胞氧化损伤及其分子机制进行了研究。结果显示，CdCl2处理显著抑制了猪胎盘滋养层细胞的活性，并激发活性氧（ROS）产生。进一步的研究表明，过量的ROS能够攻击DNA和线粒体，造成明显的DNA损伤，线粒体肿胀、嵴破坏或消失，同时，线粒体膜电位(Δψm)显著降低。CdCl2处理还将猪胎盘滋养层细胞的细胞周期阻滞在G0/G1期，并最终引起细胞凋亡。在相同条件下，CdCl2处理通过上调Bax表达、下调Bcl-2表达来提高Bax/Bcl-2比值，激活凋亡执行分子caspase-3，不可逆转地诱导猪胎盘滋养层细胞发生凋亡，并且，CdCl2处理后，MAPK信号通路中p38-MAPK显著上调。此外，动物实验表明，CdCl2可以特异性的诱导胎盘组织中滋养层细胞发生DNA损伤和细胞凋亡，但对其他细胞无明显的毒性损伤。综上所述，Cd能够通过激发氧化应激和线粒体损伤选择性地诱导胎盘组织中滋养细胞发生凋亡，这些发现有助于阐明Cd引起妊娠期胎盘损伤的细胞分子机制，并为防治危害家畜繁殖和畜牧业发展的Cd中毒问题提供理论指导。