藻蓝蛋白活性肽筛选及其对生殖细胞RIP1/RIP3/MLKL信号通路的免疫调控研究

In recent years, the incidence of infertility is on the rise, but the effective treatment is lack. It is a hot spot of research to look for natural drugs with small side effects and reproduction-improving performance from ocean. In our previous studies, we found that C-phycocyanin (C-PC) isolated form Spirulina had the function of enhancing the activities of germ cells and inhibiting cell apoptosis. However, the molecular mechanism was not clear. In addition, because of the large molecular weight of C-PC, it is not easy to pass through the cell membrane which limited its application. Our research aims at the screening of C-phycocyanin peptides with small molecular weight, high purity, easy absorbance by body and high reproduction-promoting efficacy by proteolysis, gel chromatography and mass spectrum. We will use some advanced techniques such as mouse living imaging, gene chip, RT-PCR and so on, from gene and molecular levels, to investigate the inhibitory effects of the bioactive peptides on RIP1-RIP3-MLKL apoptosis signal pathway in sperm cells and granular cells by animal and clinic experiments. In addition, from the perspective of reproductive immune regulation, we will study the regulatory effects of C-PC bioactive peptides on the activities of dNK cells in early decidua and Treg cells, and the expression of intercellular adhesion molecules, which contribute to the provide a good microenvironment for the implantation of fertilized eggs and the development of embryos. Our study will provide a new idea for the diagnosis and treatment of adverse pregnancy outcomes, supply theoretical support for the development and utilization of C-phycocyanin in the field of reproductive immunology, and has good prospect of clinical application.

近年来不孕不育发生率呈上升趋势，但缺乏有效治疗手段。从海洋中寻找改善生殖性能且毒副作用小的天然药物成为关注的热点。本课题组前期研究发现螺旋藻藻蓝蛋白(C-phycocyanin,C-PC)具有增强生殖细胞活性、抑制细胞凋亡的功能，但机制不清，且因C-PC分子量大、不易穿膜故应用受限。本研究旨在通过酶解、凝胶层析和质谱分离筛选纯度高、易吸收、促生殖功效显著的C-PC小分子活性肽。进一步采用小鼠活体成像、基因芯片、RT-PCR等技术，通过动物、临床试验从基因、分子水平探讨该活性肽对生殖细胞和颗粒细胞RIP1-RIP3-MLKL凋亡信号通路的阻抑效应，并证实其对早期妊娠蜕膜dNK细胞、Treg细胞活性及细胞间粘附分子表达的调控作用，从而为受精卵着床及胚胎发育提供良好的微环境，为不良妊娠结局的诊治提供新思路。该研究为C-PC在生殖免疫领域的开发利用提供了理论依据，具有较好的临床应用前景

本课题组前期研究发现螺旋藻藻蓝蛋白（C-phycocyanin, C-PC）具有增强生殖细胞活性、抑制细胞凋亡的功能，但机制不清，且因藻蓝蛋白分子量大、不易穿膜故应用受限。本研究旨在通过酶解、凝胶层析和质谱分离筛选纯度高、易吸收、活性高的藻蓝蛋白小分子活性肽。首先通过体外和体内实验证明了藻蓝蛋白对于生殖细胞和小鼠的生殖系统具有一定的保护作用，可以通过抑制生殖系统RIPK1-RIPK3-MLKL坏死性凋亡信号通路的激活从而使小鼠的生精能力和生育能力得到保障。然后通过基因工程技术获得高纯的藻蓝蛋白亚基，证实了藻蓝蛋白亚基可以通过抑制氧化应激的发生对生殖细胞起到保护作用，并揭示了其保护机制。通过多重生物学方法，从藻蓝蛋白酶解产物中分离纯化得到活性较好的多肽，采用斑马鱼模型评价其多肽活性，证明了藻蓝蛋白多肽可以通过抑制ROS和MDA生成，上调SOD和CAT活性从而保护组织免受氧化损伤，通过激活Nrf2信号通路发挥抗氧化作用。该多肽可作为强有力的抗氧化剂，用于预防和治疗与氧化应激相关的各种疾病。该研究为藻蓝蛋白及其多肽在生殖免疫学领域的研发利用提供实验依据该研究，具有较好的临床应用前景。. 项目执行期间发表相关论文18篇，其中14篇被SCI收录，获批国家发明专利1项，申请2项。培养博士生3名，硕士生5名，多人获得国家奖学金、省级或校级优秀毕业生、优秀毕业论文。依托项目支撑，李冰获“山东省泰山学者青年专家”称号、青岛市青年科技奖获得者，并获得山东省科技进步二等奖、山东省医学科技奖三等奖。曾在第18届国际免疫学大会、第二届中日韩青年医师论坛、山东省泰山科技论坛、济宁医学院学术论谈等分享研究成果。