通过蛋白组转录组和基因筛选研究镉诱导生物毒性的机理

Cadmium is a nonessential divalent heavy metal ion that causes toxicity in most organisms. The uptake of cd2+ is suggested to be mediated by the transporters and receptors for those essential metals such as Fe2+, Ca2+, Zn2+ or Mn2+. Cadmium tends to be accumulated in many organs and it binds to many bio-molecules particularly those with a lot of sulfhydryl groups which underlies the toxicity mechanism of cadmium. Although cd2+ is not a Fenton metal and therefore can not exert redox reactions in biological systems, it induces the formation of reactive oxygen species (ROS) indirectly by a number of ways such as depletion of redox scavengers GSH, inhibition of antioxidative enzymes and increase of labile iron pool. The association of cadmium with protein may modulate the biological function of that molecule and therefore activate or inhibit a variety of transcription factors also interfere the normal cellular signal transduction pathways. So cadmium can change the normal cellular metabolism pathway and cause cell damage. Cadmium is excreted from urine after the epithelial cells of the proximal tubule are damaged by the accumulated cadmium. The protein associated with such cell damage in urine is used as biomarker to diagnose cadmium nephrotoxicity. Recently, cd2+ was shown to interfere the function of an iron chaperone PCBP1 and the binding of cd2+ inhibits the function of PCBP1 to bind polyC single strand DNA. In this research proposal we plan to identify early biomarker for cadmium biotoxicity through proteomics approach (analysis of the cd2+ induced proteome change). The cadmium biomarker from silkworm could be used to monitor environment pollution. We also propose to study the mechanism of cadmium induced toxicity in different organs through the combination of proteomics methods and transcritomics methods. We are also going to study the mechanism of cd2+ interfering iron metabolism mediated by PCBP1 and to study the role of DMT-1 or ZIP in cd2+ uptake by cell line originated from lung. Finally, we are going to do genetic screen for cd2+ toxicity resistant genes and study the cellular function of corresponding gene products.

镉是生物非必需有毒重金属元素，其能够通过必需元素如Fe，Ca，Zn和Mn的吸收系统进入机体，并在体内积累，能紧密结合含多个巯基的蛋白或化合物。镉自身不能催化产生自由基，然而镉通过多种途径间接产生活性氧（如中和还原性化合物谷胱甘肽、抑制SOD和过氧化氢酶活性、影响铁代谢增加游离态铁等）。镉结合蛋白能改变其活性，激活或抑制多种转录因子和细胞信号传导分子，改变细胞代谢途径，损伤细胞。诊断镉中毒的尿镉或尿中的标记蛋白在肾小管上皮细胞损伤后才出现。最近，我们发现镉能结合铁分子伴侣PCBP1并抑制其结合多聚胞嘧啶序列的活性。本项目拟用蛋白组学方法筛选检测早期镉中毒的生物标记蛋白，配合基因转录组方法确定镉在不同组织中引起毒性的主要途径；研究镉通过铁分子伴侣PCBP1影响细胞铁代谢的机制和途径；确定DMT-1和ZIP蛋白在肺组织细胞中吸收镉的作用；筛选能够减轻镉的细胞毒性的基因，并研究这些基因产物的功能。

镉是生物非必需且有毒的金属元素，它广泛存在人类生活的环境中比如水、空气、土壤以及食物。环境中的镉主要是来源于采矿、电镀等工业活动。镉在人体内半衰期长达15至20年，对人体许多器官和组织有长期毒性，包括肾、肝、肺、胰腺、睾丸、骨和胎盘等，镉的体内暴露可增加这些组织器官的癌变几率。本课题用蛋白组学和基因芯片方法研究了镉暴露诱导生物标志蛋白：镉暴露导致小鼠血浆、肾脏蛋白质的差异表达，以及脾脏基因的差异表达，镉暴露导致结肠癌细胞恶性转变的差异蛋白；鉴定环境镉暴露诱导的家蚕蛋白的差异表达；研究了钙离子拮抗镉细胞毒性的分子机制。.我们发现镉暴露导致ICR小鼠血浆中13个蛋白表达发生变化，并发现一组载脂蛋白依赖镉剂量被诱导，包括载脂蛋白A-Ⅳ，妊娠带蛋白，载脂蛋白A-Ⅰ前体，视黄醇结合蛋白，淀粉体P成分前体，载脂蛋白E，载脂蛋白C-Ⅲ 前体，载脂蛋白 A-Ⅱ。血浆中胆固醇也显著增加，这不仅是由于胆固醇的摄取增加，而且是由于镉暴露导致胆固醇从肝脏和脂肪重新分布到血浆中。其分子机制是细胞的胆固醇排出通路增强（ABCA1表达增加，其抑制蛋白OSBP1稳定性下降）。.镉暴露导致肾脏组织15个蛋白上调，19个下调，参与多种代谢和调控途径，包括能量代谢，细胞信号传导，免疫调节等。发现镉暴露后细胞迁移增加，其机制在进一步研究中。发现镉破坏钙离子稳态，钙离子敏感受体CaSR激动剂能抑制镉诱导细胞凋亡，这是通过JNK和p38 MAPK信号通路介导的。GTPase信号通路参与了镉暴露导致的结肠癌恶性转变。.基因芯片分析镉暴露小鼠的脾脏发现了6个差异表达基因，其中vmp1参与细胞自噬。镉暴露导致ramos细胞自噬的增强（LC3-Ⅱ增加，P62减少），这依赖于vmp1。.目前已经相关论文4篇，获得专利一项，5篇涵盖上述主要研究结果的论文还在撰写投稿之中，预计1-2年内会发表。