Pten基因对奶牛乳腺上皮细胞泌乳的调节功能

王卓然　王春梅　王杰　李庆章　高学军

摘 要：该研究以泌乳中期的奶牛乳腺上皮细胞为模型，探索Pten基因的表达与奶牛乳腺发育和泌乳之间的关系，目的在于揭示Pten基因在奶牛乳腺中的调节作用，为动物乳腺发育和泌乳调节机制的研究提供基础资料。该研究以中国荷斯坦奶牛作为实验动物，应用荧光定量qRT-PCR、Westernblotting和免疫组织化学技术，对奶牛不同发育阶段及不同乳品质的乳腺组织中Pten mRNA和蛋白质的相对表达量进行检测；以体外培养的泌乳中期奶牛乳腺上皮细胞为研究对象，构建重组质粒pGCMV-Pten-IRES-EGFP，对细胞进行瞬时转染，进行Pten基因过表达实验；应用RNA干扰的方法用Pten siRNA瞬时转染细胞，进行Pten基因抑制实验。分别用相关试剂盒检测Pten基因过表达和抑制之后细胞？-酪蛋白、甘油三酯以及乳糖分泌的情况，为了检测Pten基因对奶牛乳腺上皮细胞活力和增殖能力的影响，分别应用CASY-TT细胞分析仪和流式细胞仪检测细胞活性和细胞周期，采用荧光定量qRT-PCR和Western blotting技术，在mRNA和蛋白水平检测与泌乳相关的信号通路基因的表达变化；同时，添加外源性催乳素和葡萄糖培养细胞，检测培养液上清中？-酪蛋白、甘油三酯和乳糖的浓度，以及Pten基因的表达量变化，从而探索Pten基因在催乳素诱导的葡萄糖转化生成乳糖过程中的作用。研究结果表明，泌乳期Pten基因表达量显著低于干乳期。与泌乳期低乳品质奶牛乳腺相比，泌乳期高乳品质的奶牛乳腺组织中Pten mRNA和蛋白表达水平分别降低了30%和40%；Pten基因过表达可抑制奶牛乳腺上皮细胞的活力、增殖能力以及？-酪蛋白、甘油三酯和乳糖的分泌量（P<0.05），使奶牛乳腺上皮细胞中MAPK、CyclinD1、AKT、mTOR、S6K1、STAT5、SREBP1、PPAR？、PRLR、GLUT1的表达下调（P<0.05），并上调4EBP1的表达水平（P<0.05）；Pten基因抑制实验表现出相反的结果；而Pten基因的过表达和抑制对ELF5的表达均无显著影响（P>0.05）。催乳素的添加能促进细胞分泌？-酪蛋白、甘油三酯和乳糖（P<0.05），并下调Pten基因表达量（P<0.05）；葡萄糖的添加显著增加了？-酪蛋白和乳糖分泌量（P<0.05），但甘油三酯含量无显著变化（P>0.05），同时Pten基因的表达量无明显改变（P>0.05）。综上所述，Pten基因参与调节奶牛乳腺上皮细胞泌乳的过程，负向调节细胞的活力、增殖能力和细胞周期，并能抑制奶牛乳腺上皮细胞分泌？-酪蛋白、甘油三酯和乳糖；这种调节作用是通过Pten基因靶向调节PI3K-AKT信号通路，进而调节其他泌乳相关信号通路基因的表达而实现的；同时发现Pten基因的表达受催乳素的负调节，但葡萄糖对Pten基因的表达水平无显著影响。

关键词：奶牛乳腺 发育 泌乳 Pten

Abstract：In the aim of detectting the role of Pten gene in the mammary gland of dairy cow， dairy cows mammary epithelial cells （DCMECs） in mid-lactation period were used as models to investigate the relationship of Pten expression and mammary glands development and lactation， which provides basic data for the study of ruminant mammary gland development and lactation mechanisms， and the theoretical support for milk production and milk quality of the artificial regulation at the same time. In this research， Holstein dairy cows were used as experimental animals， applying to qRT-PCR， Western blotting， and immunofluorescence triple staining technology， Pten mRNA and protein expression at different development stages and various milk qualities of dairy cows mammary gland tissue were detected. Furthermore， DCMECs as research objects in vitro were used to study the function of Pten gene. Recombinant plasmid pGCMV-Pten-IRES-EGFP was constructed and transient transfected into cells to prosue the Pten gene overexpression experiment. Meanwhile， RNAi method was used to transfect Pten siRNA in the Pten gene inhibition experiment. We determined concentrations of β-casein， triglyceride， and lactose following Pten gene overexpression and inhibition by specific kits. To determine whether Pten gene affected DCMEC viability and proliferation， cells were analyzed by CASY-TT and flow cytometry. Genes involved in lactation-related signaling pathways were detected by qRT-PCR and Western blotting. After prolactin and glucose were added to the cell cultures， concentrations of β-casein， triglyceride， and lactose were detected， and Pten gene expression was also assessed. Thus investigating the role of Pten gene in the process of glucose transform into lactose induced by prolactin. In summary， we showed that Pten gene is specifically involved in lactation of dairy cow mammary epithelial cells， and down-regulates DCMEC viability， proliferation ability， and the cell cycle along with β-casein， triglyceride， and lactose secretion. Pten gene targets and regulates the PI3K/AKT pathway， which in turn regulates other lactation-related signaling genes. Moreover， the expression of Pten gene can be down-regulated by prolactin， but the introduction of glucose to culture medium revealed no significant difference in Pten gene expression level in DCMECs.

Key Words：Dairy cow mammary gland；Development；Lactation；Pten

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