硫化氢中毒生物标志物的法医毒物分析筛选及相关机理研究

Hydrogen sulfide (H2S) is a toxic gas that has been caught high attention in the field of forensic toxicology. H2S poisoning is familiar and the mortality rate is high. A detection of the internal indicators is the basic of evaluation for H2S poisoning and moreover it is a challenging study. In the previous study of Sprague-Dawley rats, we found that the levels of protein expression associated with the mechanism of H2S poisoning had significant changes in the serum and lung tissue. The result may prompt the relationship between the changes and the selection of biomarkers of H2S poisoning. To ensure the accuracy and reliability of the screening results, we use H2S poisoning cadavers as objects on the basic of previous studies. Firstly, we screen the specific expressed-protein of H2S poisoning under the technology of quantitative proteomics coupled with bioinformatics. Secondly, we use molecular biotechnology and numbers of samples to screen and verify the specific expressed-protein, and then we obtain the potential biomarkers of H2S poisoning. Lastly, we analyze the biological function of the potential biomarkers on cellular level, and explore the mechanism of H2S poisoning. The results of this project will help to solve the current bottleneck problem of estimating H2S poisoning, to clarify the molecular mechanism of H2S poisoning, and to promote the value of forensic toxicological evidence.

硫化氢中毒较为常见且致死率高，是法医毒物学领域关注度较高的毒性气体。硫化氢中毒体内目标物的检测是评判硫化氢中毒的主要依据，这也是困扰硫化氢中毒评判的难点问题。申请者在动物实验研究中发现，受硫化氢暴露的SD大鼠血清和肺组织中，诸多与硫化氢中毒机理相关的蛋白质表达水平存在显著变化，这提示硫化氢中毒生物标志物的筛选，可能是解决硫化氢中毒评判问题的有效方法。为保证人体硫化氢中毒生物标志物筛选结果准确可靠，申请者在前期研究基础上，提出以硫化氢中毒尸体为研究对象，综合运用定量蛋白质组学结合生物信息学技术，筛选硫化氢中毒特异性差异表达蛋白质，运用分子生物学技术通过大样本筛选和验证，获得若干硫化氢中毒潜在生物标志物，并从细胞水平解析硫化氢中毒生物标志物的生物学功能，探索硫化氢中毒机理。项目的实施有助于解决目前硫化氢中毒评判的瓶颈问题，阐明硫化氢中毒的分子机理，提高毒物司法鉴定的证据价值。

本研究项目以高（400ppm）、中（200ppm）、低（100ppm）四个浓度的硫化氢（H2S）暴露环境中ICR小鼠为实验对象，提取受试小鼠血液、脑组织和肺组织，通过非靶向液相色谱-串联质谱检测方法，遴选H2S中毒小鼠体内差异蛋白，并通过生物信息学技术和生物学方法对差异蛋白进行生物学验证，进一步探讨H2S中毒的毒理学机制，筛选硫化氢特异性蛋白。.通过HE染色实验、凋亡实验和WB实验发现H2S处理能够引起脑和肺组织损伤、细胞凋亡并确定模型构建成功；质谱实验结果发现H2S处理后Lamb1表达降低、C9表达升高，又通过WB验证Lamb1和C9表达从而验证质谱分析结果；最后细胞水平证明H2S以PARP1 / BAX，C9和Lamb1依赖性方式促进脑和肺细胞损伤的机制。一方面，过量的H2S通过上调PARP1 / BAX和C9促进小鼠脑细胞和肺细胞的凋亡和炎症。另一方面，过量的H2S通过下调Lamb1促进小鼠脑细胞和肺细胞的纤维化。