Ca2+信号通路介导猪骨髓MSCs成脂分化的分子机制及其营养调控

Adipocytes are derived from multipotential mesenchymal stem cells (MSCs). Studying the committed differentiation from MSCs to adipocytes and its regulation mechanism has important significance on improving carcass traits and pork quality in livestock. Recent studies show that Ca2+ signaling plays an important role in the differentiation of preadipocytes. However, the effects and its mechanism of Ca2+ signaling on the committed differentiation to adipocytes of porcine bone marrow MSCs remain unclear. Thus, by using the model of adipogenic differentiation of porcine porcine bone marrow MSCs we previously constructed, we firstly examine the expression pattern of molecules involved in Ca2+ signaling. Meanwhile, Ca2+ channel agonists, blocker and upstream regulating agents were used to investigate the effects of Ca2+ signaling on the committed/adipogenic differentiation of porcine bone marrow MSCs. Furthermore, the techniques including overexpression and interference, chromatin immunoprecipitation assay (CHIP) were applied to explore the molecular mechanism involved in Ca2+ signaling mediated adipogenic differentiation of porcine bone marrow MSCs. In vivo study, we investigate the role and mechanism of the regulating agents of Ca2+ signaling (Nifedipine and calcium formate) in carcass traits and pork quality of pigs. The results of the present project can not only reveal the molecular mechanism involved in Ca2+ signaling mediated adipogenic differentiation of porcine bone marrow MSCs, but also provide theoretical and experimental basis in the nutrition regulation technology to improve porcine carcass traits and pork quality.

脂肪细胞来源于多潜能间充质干细胞（MSCs），研究MSCs向脂肪细胞定向分化及其调控机制对提高家畜胴体品质和肉品质具有重要意义。研究表明，Ca2+信号对脂肪前体细胞分化聚酯具有重要作用，但其对MSCs定向分化为脂肪细胞的作用及机制尚不清楚。本项目利用前期建立的猪骨髓MSCs成脂分化模型，首先观察猪骨髓MSCs成脂分化过程中Ca2+信号相关分子表达模式，其次应用Ca2+通道激动剂、阻断剂及其上游调控物，研究其对猪骨髓MSCs成脂分化的作用，进而采用过表达和干扰、染色质免疫共沉淀等技术，深入揭示Ca2+信号调控猪骨髓MSCs成脂分化的分子机制。在活体水平，研究Ca2+信号调控物（Nifedipine和甲酸钙）对猪胴体品质和肉品质的影响及其机制。项目研究结果不仅能深入揭示Ca2+信号通路介导猪骨髓MSCs成脂分化的分子机制，同时可为以改善猪胴体品质和肉品质为目标的营养调控技术提供理论与试验依据。

研究间充质干细胞（MSCs）向脂肪细胞定向分化及其调控机制对提高家畜胴体品质和肉品质具有重要意义。本项目利用猪骨髓MSCs（pBMSCs）成脂分化模型，首先检测了pBMSCs成脂分化过程中Ca2+信号相关分子表达模式，结果表明，在pBMSCs成脂分化第5天，[Ca2+]o显著促进细胞膜上钙离子通道、CaSR以及成脂定向相关基因的mRNA表达，提示第5天是pBMSCs成脂定向的关键时期。.其次，我们研究了Ca2+信号对pMSCs增殖和成脂分化的作用及其分子机制。pBMSCs增殖结果显示，[Ca2+]o能够通过激活pBMSCs细胞膜上的CaSR，使细胞内钙离子浓度（[Ca2+]i）升高，升高的[Ca2+]i通过激活ERK信号通路进一步促进pBMSCs增殖。在pBMSCs成脂分化方面，[Ca2+]o通过VGCC和CaSR升高pBMSCs中的[Ca2+]i浓度，随后激活下游的CaMKII信号通路，并通过加强胰岛素下游的PI3K/Akt-FoxO1-PPARγ信号通路，最终促进pBMSCs的成脂分化；同时，[Ca2+]o能够通过激活CaMKII-PI3K/Akt-AS160通路增强pBMSCs成脂过程中细胞GLUT4转位及葡萄糖吸收。.最后，我们研究了Ca2+信号对小鼠脂肪沉积和糖代谢、猪胴体品质和肉品质的影响及机制。结果表明，饮水添加氯化钙可以降低高脂日粮饲喂小鼠体内脂肪含量，但增加皮下脂肪细胞的数量，改善葡萄糖稳态；育肥猪饲喂甲酸钙对其生长性能（体增重、饲料转化率）、胴体品质（背膘厚、眼肌面积）和肉品质（pH值、肉色）无显著影响，但却提高了肉的滴水损失。此外，甲酸钙可减小皮下脂肪细胞的平均直径，增加脂肪细胞的数量，同时激活皮下脂肪中PI3K/Akt信号通路和促进成脂标志基因表达。.总之，本项目研究结果表明，钙离子通过CaSR-[Ca2+]i-ERK信号通路促进pBMSCs增殖，通过VGCC-[Ca2+]i-CaMKII-PI3K/Akt信号通路促进pBMSCs成脂分化及对葡萄糖的吸收，且钙离子对pBMSCs成脂分化的促进作用主要发挥在成脂定向阶段。在活体上，钙离子可通过PI3K/Akt信号通路促进脂肪组织细胞数量的增加。研究结果揭示了Ca2+信号通路介导pBMSCs成脂分化的分子机制，为改善猪胴体品质和肉品质的营养调控提供了一定的理论基础和技术支撑。