基于miRNA-mRNA-protein多生物学指标检测模式构建皮肤损伤时间推断体系的研究

Due to certain key problems interfering with skin wound age estimation in the world-wide medico-legal practice and in the light of miRNA function and its properties, The present project focuses on establishment of a novel assay mode of miRNA-mRNA-protein, the “functional unit” pattern that we term for determination of the functionally-related multiple biomarkers with molecular biological technique and bioinformatic analysis. By “functional unit” assay mode, expressions of miRNAs, its target gene (mRNA) and proteins are determined at different posttraumatic intervals in the skin. Morphological analyses are conducted by histochemical and molecular pathological procedures. The multiple biomarkers are investigated and evaluated in the injured skin specimens with different postmortem changes in the animal model. The multiple biomarkers are assayed with validation and verification in the human injured skin samples collected from the deceased under relevant conditions. A system for skin wound age estimation including hierarchical biomarkers, data bank and mathematic models with techincal method will be developed in skin specimens with or without postmortem changes, by which skin wound age estimation will tend to be more accurate in order to realize “precision medico-legal skin wound age estimation”, and postmortem change, a conundrum challenging the skin wound age estimation is expected to be solved. Also, the project would provide basis for translation from the experimental investigation to the medico-legal practice for the skin wound age estimation.

基于国内外法医学皮肤损伤时间推断中存在的关键性问题以及miRNA的功能和特性，本项目通过小鼠皮肤切创模型，采用分子生物学、生物信息学技术建立miRNA-mRNA-protein“功能组”检测新模式，进行功能关系密切的多生物学指标检测。以“功能组”检测模式，对不同损伤时间段皮肤组织中miRNAs、及其靶基因和蛋白的表达进行检验；采用组织化学、分子病理学技术进行形态学分析，并进行不同程度死后变化对损伤皮肤的多生物学指标进行检验、评价。同时，应用相应影响因素条件下的人尸体皮肤损伤样本检材进行检验、验证，构建基于新鲜及不同程度死后变化条件下包括损伤时间推断指标构成、数据库、数学模型及技术方法等项目的皮肤损伤时间推断体系，为提高皮肤损伤时间推断的准确性，实现“精准法医学皮肤损伤时间推断”目标，并解决尸体死后变化影响法医学实践中损伤时间推断的“瓶颈”问题以及成果转化提供依据。

皮肤损伤时间推断是法医病理学实践中的一项重要工作。本项目采用分子生物学、生物信息学技术，对不同损伤时间段皮肤组织中miRNAs、及其靶基因和蛋白的表达进行检验， 建立miRNA-mRNA-protein“功能组”检测新模式。通过高通量测序检测并分析皮肤损伤后差异性表达的miRNA与mRNA，识别出由150个miRNA和763个mRNA形成的1573对miRNA-mRNA靶向调控网络。对差异表达miRNA的靶基因进行GO及KEGG富集分析结果显示，大多数基因富集到参与连接、酶活性、分子转导活性调控等分子功能。进一步通过荧光素酶报告基因实验验证，及蛋白表达水平检测，筛选出以“mmu-miR-15b-5p/Rsad2”、“mmu-miR-31-5p/Cst6”和“mmu-miR-1983/Krt77”为代表的“功能组”指标数据集。随后经数学模型构建及模型验证，发现该方法在伤后13天内均具有良好的损伤时间推断准确率。本项目为提高皮肤损伤时间推断的准确性，实现“精准法医学皮肤损伤时间推断”的目标提供了新的依据和前进方向。