GPR39-PI3K/Akt通路调控猪肌内脂肪前体细胞增殖与分化的机制研究

Pig intramuscular preadipocyte proliferation and differentiation directly affect the content of intramuscular fat, further to influnce on meat quality. To present, the regulatory mechanisms of pig intramuscular preadipocyte proliferation and differentiation are uncertain, so the research of new regulatory genes has important scientific significance in improving meat quality. It has been proved that GPR39 could meditate signaling pathways to participate in the process of tissue regeneration and cell differentiation, which might be a key candidate gene affecting fat deposition. Therefore, based on the previous studies we have done, using pig intramuscular preadipocytes cultured in vitro as a model, the cells were treated with the methods of overexpression and blocking. Cell proliferation and viability were analyzed by MTT assay, cell apoptosis was measured by Fluorescence Activating Cell Sorter, the degree of differentiation and adipogenisis were studied by Oil Red O staining extraction assay, mRNA expression of PPARγ, GSK3, Caspase9,C/EBPβ and ADD1 genes was detected by Real-Time quantitative PCR, and protein expression of PI3K, PDK1 and Akt was measured by Western Blot. It aims to reveal the regulatory mechanisms of GPR39-PI3K/Akt signaling pathwayin pig intramuscular preadipocyte proliferation and differentiation, providing the knowledge-based theory for developing the new technology to improve pork quality.

猪肌内脂肪前体细胞增殖分化直接影响肌内脂肪含量，进而影响肉品质。目前，肌内脂肪前体细胞的调控机制仍未完全揭示，已成为制约肉质改良的难题，故研究新的调控基因具有重要的科学意义。已证实，GPR39可介导信号通路参与组织再生和细胞分化等过程，可能是影响猪肌内脂肪沉积的一个关键候选基因。为此，本项目结合前期工作，拟以猪肌内脂肪前体细胞为模型，采用过表达法与阻断法处理体外培养的猪肌内脂肪前体细胞，通过MTT 比色法检测细胞活性及增殖状况，流式细胞仪进行凋亡定量分析，油红O染色化学比色法观察细胞内脂肪生成及细胞分化程度，实时荧光定量RT-PCR法分析PPARγ、GSK3、Casp9、C/EBPβ与ADD1等基因mRNA的表达，Western blot 检测PI3K、PDK1与Akt，初步揭示GPR39-PI3K/Akt通路对猪肌内脂肪前体细胞增殖分化的调控机制，为改善猪肉品质的新技术奠定理论基础。

据研究，GPR39可能是调控猪肌内脂肪沉积的候选基因。为此，本项目采用MTT法、RT-PCR、Western Blot、siRNA干扰等方法，分析猪肌内前体脂肪细胞GPR39-PI3K/Akt通路在肌内脂肪沉积中的作用机制。初步揭示：1）30 d，梅花猪背最长肌脂肪含量、脂肪细胞数目与大小均低于长白猪；60 d后，其肌内脂肪含量、脂肪细胞数目与大小显著高于长白猪；机理为前者HSL与HMGCoA mRNA表达低于后者，而Adiponectin表达二者差异不显著。2）采用无血清培养体系培养猪肌内前体脂肪细胞，利用GPR39过表达，以PI3K抑制剂LY294002及AKT抑制剂PCTC阻断PI3K/Akt通路，发现GPR39 mRNA和蛋白均有表达，且Zn2+诱导可明显上调GPR39水平。GPR39过表达可提高细胞OD值，上调PPARγ、C/EBPα、ADD1 mRNA表达和pGSK3蛋白表达，但抑制了Caspase-9 mRNA表达。此外，GPR39对PI3K/Akt通路上的因子PI3K、PDK1与总Akt表达无影响，但可上调pAkt而活化该信号通路。当加入PDTC和LY294002抑制剂后，GPR39诱导的猪肌内脂肪前体细胞的增殖与分化明显受阻。证实GPR39是Zn2+传感器，可通过PI3K/Akt细胞信号通路促进猪肌内前体脂肪细胞增殖和分化。3）体外培养猪皮下脂肪前体细胞，经不同浓度Obestatin处理，对前体脂肪细胞GPR39表达无影响，但可通过上调PPARγ和C/EBPα mRNA表达，促进细胞增殖和分化，并通过降低Caspase-3、7与9表达，抑制细胞凋亡。4）采用两步胶原酶灌注法成功获得猪肝细胞，用不同剂量GPR39处理后发现，明显促进细胞增殖，显著降低了细胞甘油三酯含量与HMG-CoA表达，上调脂质代谢相关因子Apo-AI、ACC-α与HSL表达，且具剂量依赖性，提示GPR39同样可调节猪肝细胞增殖和脂代谢。以上研究成果为猪健康养殖及改善猪肉品质的新技术奠定理论基础。