胶原XV通过“Ca2+-内质网”轴调节猪白色脂肪棕色化的作用机制

Collagen XV is the important components of the extracellular matrix in adipocytes, which have been confirmed closely related to white adipocytes differentiation and lipid metabolism. Our group further finds that the browning of the white adipose exists in pig. And the ColXV expression can be significantly induced by cold stimulation in piglet subcutaneous and visceral white adipose, indicating ColXV may take part in the regulation of white adipose browning, but the molecular mechanism is still unclear. In this study, Guanzhong black pigs are used as the experimental animals. Through qRT-PCR, Western Blotting, fluorescence probe, laser scanning confocal, patch clamp gene chip technology and other technics are applied. We investigate the expression pattern of ColXV during white adipocyte browning in pig, as well as the change of intracellular Ca2+ concentration, resolve the regulating effect of ColXV on white adipocytes browning in pig and filtrating the key genes, the significant signaling pathway, explore the relationship between Ca2+signal and the regulation of ColXV on browning, reveal the important role of "Ca2+-ER" axis on the regulation of ColXV on adipocyte browning in pig, and explain the transcription regulation of ColXV and its effect on adipocyte browning in pig. Results would lay a theoretical basis for further study of the genetic manipulation of white adipose browning in pig，the reduction of fat deposition in pigs and the increase of lean meat percentage. It also could offer a new strategy for the prevention and treatment of human dyslipidemia, obesity and related diseases.

胶原XV（Collagen XV）是脂肪细胞外基质的重要组分，并与白色脂肪细胞分化和脂质代谢密切相关。本课题组已发现猪体内存在白脂棕色化现象，且在冷刺激的仔猪皮下和内脏白脂中ColXV表达显著提高，提示其可能参与白脂棕色化作用，但具体作用及机制尚不清楚。本项目拟以关中黑猪为试验对象，通过实时定量PCR、蛋白杂交、荧光探针、激光共聚焦显微镜、膜片钳、基因芯片等技术，探索猪白脂棕色化中ColXV表达规律及Ca2+浓度变化，研究ColXV对猪白脂棕色化的调节作用并筛选关键基因及相关信号通路，探讨Ca2+信号对ColXV调节猪白脂棕色化的影响，揭示“Ca2+-内质网”轴在ColXV调节猪白脂棕色化中的作用，探明ColXV转录因子及其对猪白脂棕色化的调节作用；为深入研究猪白脂棕色化的遗传调控、降低猪脂肪沉积、提高瘦肉率提供理论依据，为防治人类脂代谢紊乱、肥胖症等疾病提供新思路。

以关中黑猪作为试验动物，应用组织RNA提取及脂肪细胞培养、Real-time PCR、Western Blot ting、免疫荧光染色和iRNA干扰等研究技术，构建 ColXV 的超表达载体和干扰载体, 与猪脂肪细胞共培养、转染，检测脂肪细胞分化及棕色化标志基因，脂代谢关键酶的表达量及磷酸化水平。探讨 ColXV 对猪脂肪细胞分化、脂肪酸氧化和白色脂肪棕色化的影响，阐释 ColXV 通过“Ca2+-内质网”轴调控脂肪细胞分化及棕色化的分子机理，初步揭示ColXV通过“Ca2+-内质网”轴介导猪脂肪沉积和代谢的机制。这些结果将对进一步阐明猪脂肪组织ColXV 对脂肪组织重塑及白脂棕色化的调控机制，揭示ColXV 对脂肪细胞内质网应激及白脂棕色化的作用机理，最终控制猪脂肪沉积，提高瘦肉率具有重要的理论价值和实践意义，并对预防和治疗人类肥胖及其相关疾病具有一定的参考价值。