丙酮酸激酶M2对奶牛乳腺上皮细胞增殖和乳合成的影响

Glucose plays a pivotal role in mammary gland development and lactation. Higher amount of glucose flows into pentose phosphate pathway than into tricarboxylic acid cycle in mammary gland. This physiological phenomenon may be associated with one type of the rate-limiting enzyme of glycolysis, pyruvate kinase M2 (PKM2). Previous research has suggested that, the endocrine hormones affect proliferation and milk synthesis of mammary epithelial cells maybe through its regulation of PKM2. By utilizing biochemistry and molecular biology technologies, the relevance of PKM2 enzymatic activity with its expression, phosphorylation and oligomer forms will be examined in bovine mammary gland in different developmental stages. Next, the mechanisms of PKM2 expression and post-translational modifications affected by endocrine hormones will be studied in bovine mammary epithelial cells. Last, the functional roles of PKM2 in proliferation and milk synthesis will be explored in PKM2 knockout or overexpressing bovine mammary epithelial cells. In this manner, our research can, not only, clarify the functions and mechanisms of PKM2-regulated cell proliferation and milk synthesis in mammary epithelial cells, but also consummate the theory of development and lactation biology of mammary gland and provide scientific guidance for the development of the dairy industry.

葡萄糖对于乳腺的发育与泌乳至关重要。奶牛乳腺中进入磷酸戊糖途径代谢的葡萄糖量远高于三羧酸循环，这一生理现象与一种糖酵解关键限速酶丙酮酸激酶M2（PKM2）活性相关。已有前期研究提示，奶牛乳腺发育过程乳腺上皮细胞增殖及乳合成可能与内分泌激素调控PKM2表达紧密关联。本申请项目利用生物化学与分子生物学方法，检测鉴定不同发育期奶牛乳腺PKM2的表达、磷酸化修饰、多聚体存在形式的差异及其与酶活性的关系；以奶牛乳腺上皮细胞为模型，揭示PKM2表达调控和翻译后修饰的内分泌激素影响机制；通过基因敲除和过表达技术，探索奶牛乳腺上皮细胞增殖和乳合成过程PKM2的功能作用。这些围绕PKM2对奶牛乳腺上皮细胞增殖和乳合成的影响研究，不仅将揭示PKM2在奶牛乳腺上皮细胞增殖和乳合成过程发挥的功能作用及相关分子机制，完善乳腺发育与泌乳生物学的理论内容，还能为乳产业的改良提供理论指导。

葡萄糖对于乳腺的发育与泌乳至关重要。奶牛乳腺中进入磷酸戊糖途径代谢的葡萄糖量远高于三羧酸循环，这一生理现象与一种糖酵解关键限速酶丙酮酸激酶M2（PKM2）活性相关。为了研究在奶牛乳腺上皮细胞MAC-T中敲低PKM2对脂质代谢的影响，我们利用慢病毒介导的shRNA构建了稳定表达低水平PKM2的MAC-T细胞。结果显示虽然敲低PKM2并不影响MAC-T细胞的增殖，但是降低PKM2的表达能够通过SREBP途径显著减少脂质合成关键酶的mRNA和蛋白水平的表达。研究结果表明，下调PKM2的表达显著影响脂质在奶牛乳腺上皮细胞MAC-T的合成。这将拓展我们对于奶牛乳腺上皮细胞中糖酵解和脂质代谢联系的理解。