小鼠皮肤角化细胞源外泌体调控毛色形成的机理研究

Coat color is a decisive factor in economic value of animals. There are many genes involved in the process of forming coat color and the regulation pathways were complex. Exosomes play an important role on information and genetic materials transmission between cells and cellsOur previous study found that many genes and miRNAs from keratinocytes can regulate the melanin formation in melanocytes. This study tries to indicate that keratinocytes release exosomes and selectively carry mRNAs, miRNAs and proteins to specifically target melanocytes and then regulate pigmentation by altering gene expression and enzyme activity. Experiment was designed to culture two kinds of keratinocytes. One is from different coat color mice and the other is irradiated by ultraviolet. We isolate exosomes, analyze and screen the predominant genetic resources associated with coat color through the transcriptomics, proteomics and miRNAs expression profile. Then the study was made to reveal the mechanism that the predominant genetic resources specifically targeted melanocytes and mediated melanin synthesis through exosomes released by keratinocytes via cell culture, immune tracer observation, cell transfection, pigment content determination, and injection exosomes derived keratinocytes into knocked-out mice skin. Through this study, we expect to explain the relationship between keratinocytes and melanocytes in the process of formation of coat color or after the ultraviolet irradiation, to explore new ideas in the mechanism of coat color formation, to lay a solid theoretical foundation for getting rich coat color resources by manual intervention in animal husbandry.

毛色是毛用动物经济价值的决定性因素，其形成涉及众多基因，调控路径复杂。开发毛色性状的基因资源是目前面临的重要问题。外泌体是重要的细胞间信息传递分子及遗传物质传递载体。我们前期研究发现来源于角化细胞的多个基因、miRNAs可调控黑色素细胞色素生成。故本研究提出小鼠角化细胞释放外泌体，选择性地携带遗传资源特异性靶向黑色素细胞调节其色素沉积。本项目通过培养不同毛色角化细胞及经紫外线照射的角化细胞，分离外泌体，经蛋白质组学、转录组学和miRNAs表达谱筛选分析与毛色形成相关的优势遗传资源；经细胞共培养、免疫示踪、细胞转染、色素含量测定、制备基因敲除鼠并活体注射等揭示优势遗传资源通过角化细胞源外泌体特异性靶向黑色素细胞介导色素合成的机制，旨在阐明毛色形成过程中或皮肤受紫外线照射后黑色素细胞与角化细胞之间的协调性，为深入研究毛色形成机理开辟新思路，为畜牧业人工干预获得丰富的毛色资源打下坚实的理论基础。

毛色是毛用动物经济价值的决定性因素，其形成涉及众多基因，调控路径复杂。开发毛色性状的基因资源是目前面临的重要问题。外泌体是重要的细胞间信息传递分子及遗传物质传递载体。结合前期研究成果，本项目通过紫外线照射角化细胞，分离外泌体，转录组学和miRNAs表达谱筛选分析、细胞共培养、免疫示踪、细胞转染、色素含量测定等方法，探寻了不同处理的角化细胞源（中波紫外线照射后小鼠角化细胞）外泌体中差异表达mRNAs、miRNAs；选取miR-330、miR-29、NOS等基因转染角化细胞探索了角化细胞源外泌体对黑色素细胞色素生成功能的影响；对有显著效果的miR-330进行了角化细胞外泌体工程化处理，并在动物水平验证了外泌体特异性靶向黑色素细胞，通过改变其毛色基因表达和酶活性来影响毛色形成的机制；并延伸性开展了外泌体在肌肉发育调控方面的部分工作。研究结果阐明了毛色形成过程中或皮肤受紫外线照射后黑色素细胞与角化细胞之间的协调性，为深入研究毛色形成机理开辟新思路，为畜牧业人工干预获得丰富的毛色资源打下坚实的理论基础。研究结果如下：.1、角化细胞受紫外线照射处理后，其分泌的外泌体包含的遗传物质发生显著变化，miR-330显著降低，NOS显著升高等；.2、黑色素细胞过表达外源性miR-330-5p后，可以下调细胞中TYR表达和黑色素产量；.3、制备的高表达miR-330-5p角化细胞源外泌体与黑色素细胞共培养后发现，外泌体可进入黑色素细胞，携带的miR-330-5p调控了黑色素细胞黑色素的生成，对黑色素细胞色素生成相关的MITF、TYR、TYRP1、TYRP2等基因产生了调控作用，TYR是miR-330-5p的直接作用靶点。.4、高表达miR-330-5p角化细胞源外泌体注射C57BL/6小鼠后发现，高表达miR-330-5p的角化细胞外泌体动物水平可影响毛色的形成；.6、试验发现，紫外线照射提高了角质形成细胞外泌体一氧化氮合成酶的活性；紫外线照射对共培养的角质形成细胞和黑色素细胞黑色素生成产生影响，外泌体携带的NOS可能发挥了重要作用。