硫化氢对烧伤大鼠表皮细胞的影响及其机制研究

Wound recovery is the fundamental problem of wound treatment after burn, however, only epidermal cells cover the wound to finalize the wound healing. Endoplasmic reticulum stress and mitochondrial damage duration and stress level has important effects on the function and survival of stressed cells, which determine the development of adaption, damage or apoptosis of stress cells. In recent years, the physiological effects of endogenous H2S in the nervous system, cardiovascular system, digestive and reproductive systems have been proved, which are increasingly attracted attention in the aspects of drug development prospects..The project selects whether hydrogen sulfide protect epidermal cells post-burn as the starting point, to detect the cells’ apoptosis, endoplasmic reticulum stress and mitochondrial damage. Hydrogen sulfide has a clear role in protecting cells, and analyze correlation of endoplasmic reticulum stress, mitochondrial injury and cells apoptosis after interfered by hydrogen sulfide; further study the signaling pathway of hydrogen sulfide protected epidermal cells. First clear whether the ATP-sensitive potassium pathway are the channels in cell membranes that hydrogen sulfide-mediated epidermal cell; then study whether the intracellular calcium is the intracellular second messenger of hydrogen sulfide signaling pathways, also analyze calcium is the factor of leading mitochondrial damage. For improving the hydrogen sulfide-mediated endoplasmic reticulum stress signaling pathways, to investigate whether miR-455 is the adjusting signal of endoplasmic reticulum stress; to measure the changes of miR-455 from animal level; cells transfected with miR-455 and interfere by hydrogen sulfide. From the transcriptional regulation to disclose possible signal transduction mechanisms of hydrogen sulfide regulated endoplasmic reticulum stress and apoptosis related genes. This project is innovative for expanding physiological and pathological study of burn epidermal cells.

烧伤后创面修复是创面治疗的根本问题，但表皮细胞覆盖创面才能最终完成愈合。内质网应激和线粒体损伤持续时间及应激水平决定应激细胞适应、损伤或凋亡的发生与发展。近年来已证明内源性硫化氢在神经系统、心血管系统、消化系统及生殖系统等方面的生理作用，其在药物开发的前景等方面也越来越引起人们的重视。.本项目从硫化氢是否保护烧伤表皮细胞为切入点，检测包括细胞凋亡，内质网应激及线粒体损伤。明确硫化氢具有保护细胞的作用，并分析硫化氢干预后内质网应激、线粒体损伤与细胞凋亡的相关性；再研究硫化氢保护烧伤表皮细胞的信号传导通路，首先明确ATP 敏感性钾通道是否为硫化氢介导表皮细胞的细胞膜通道；随后研究细胞内钙离子是不是硫化氢信号通路的胞内第二信使，同时分析钙离子也是导致线粒体损伤的因素。为完善硫化氢介导内质网应激的信号通路，探讨miR-455是否为内质网应激的调节信号，从动物水平取血测定miR-455变化，并在细胞水平转染miR-455后用硫化氢进行干预，从转录调控这一新角度揭示硫化氢调控内质网应激-凋亡相关基因的可能信号转导机制。本项目对拓展烧伤表皮细胞的病理生理研究具有创新性。

烧伤后创面修复是创面治疗的根本问题，但表皮细胞覆盖创面才能最终完成愈合。内质网应激和线粒体损伤持续时间及应激水平对于应激细胞的功能与生存均具有重要影响，决定应激细胞适应、损伤或凋亡的发生与发展，对机体免疫系统功能状态可能具有重要作用。近年来已证明内源性硫化氢在神经系统、心血管系统、消化系统及生殖系统等方面的生理作用，其在药物开发的前景等方面也越来越引起人们的重视。本课题用烧伤大鼠血清处理离体培养的大鼠表皮细胞。从信号传导入手，研究线粒体ATP敏感性钾通道和膜ATP敏感性钾通道是硫化氢介导表皮细胞的通道；随后接效应器，研究硫化氢与内质网应急与细胞凋亡和线粒体损伤的相关性，并进一步完成细胞第二信使内钙离子浓度及干预细胞凋亡miR-455的检测，探究硫化氢影响烧伤大鼠表皮细胞信号传导通路。主要研究结果：1、微量外源性硫化氢对烧伤表皮细胞有保护作用。烧伤应激下会造成大鼠表皮细胞内 H2S/CSE体系的损伤，使其效应降低；硫氢化钠使烧伤大鼠血清干预大鼠表皮细胞的凋亡率下降。2、外源性硫化氢减轻烧伤大鼠血清干预的离体培养的表皮细胞的线粒体的损伤和内质网应激。3、ATP敏感性K+通道是介导硫化氢对表皮细胞发挥病理生理作用的通路。外源性H2S通过调控KATP通道蛋白水平拮抗烧伤血清干预表皮细胞的损伤，对烧伤后表皮细胞起到保护作用。4、钙离子是硫化氢在保护表皮细胞信号通路中的第二信使。外源性 H2S 在一定时间范围内可降低烧伤应激后表皮细胞内未结合Ca2+浓度并增加烧伤应激后表皮细胞 CAM 的蛋白表达量。5、硫化氢抑制内质网应激介导细胞凋亡的机制。外源性硫化氢对烧伤大鼠体内miRNA-455水平影响的研究。烧伤大鼠体内miRNA-455含量较正常大鼠体内miRNA-455含量明显升高。浓度为150μmol /L外源性NaHS干预，可明显降低烧伤大鼠表皮细胞内miRNA-455含量，减轻了烧伤后大鼠表皮细胞内质网应激水平。为研发含有硫化氢成分的药物用于救治烧伤患者提供了基础实验数据。