王卓然　王春梅　王杰　李慶章　高學(xué)軍

摘 要：該研究以泌乳中期的奶牛乳腺上皮細(xì)胞為模型，探索Pten基因的表達(dá)與奶牛乳腺發(fā)育和泌乳之間的關(guān)系，目的在于揭示Pten基因在奶牛乳腺中的調(diào)節(jié)作用，為動物乳腺發(fā)育和泌乳調(diào)節(jié)機(jī)制的研究提供基礎(chǔ)資料。該研究以中國荷斯坦奶牛作為實(shí)驗(yàn)動物，應(yīng)用熒光定量qRT-PCR、Westernblotting和免疫組織化學(xué)技術(shù)，對奶牛不同發(fā)育階段及不同乳品質(zhì)的乳腺組織中Pten mRNA和蛋白質(zhì)的相對表達(dá)量進(jìn)行檢測；以體外培養(yǎng)的泌乳中期奶牛乳腺上皮細(xì)胞為研究對象，構(gòu)建重組質(zhì)粒pGCMV-Pten-IRES-EGFP，對細(xì)胞進(jìn)行瞬時轉(zhuǎn)染，進(jìn)行Pten基因過表達(dá)實(shí)驗(yàn)；應(yīng)用RNA干擾的方法用Pten siRNA瞬時轉(zhuǎn)染細(xì)胞，進(jìn)行Pten基因抑制實(shí)驗(yàn)。分別用相關(guān)試劑盒檢測Pten基因過表達(dá)和抑制之后細(xì)胞？-酪蛋白、甘油三酯以及乳糖分泌的情況，為了檢測Pten基因?qū)δ膛Ｈ橄偕掀ぜ?xì)胞活力和增殖能力的影響，分別應(yīng)用CASY-TT細(xì)胞分析儀和流式細(xì)胞儀檢測細(xì)胞活性和細(xì)胞周期，采用熒光定量qRT-PCR和Western blotting技術(shù)，在mRNA和蛋白水平檢測與泌乳相關(guān)的信號通路基因的表達(dá)變化；同時，添加外源性催乳素和葡萄糖培養(yǎng)細(xì)胞，檢測培養(yǎng)液上清中？-酪蛋白、甘油三酯和乳糖的濃度，以及Pten基因的表達(dá)量變化，從而探索Pten基因在催乳素誘導(dǎo)的葡萄糖轉(zhuǎn)化生成乳糖過程中的作用。研究結(jié)果表明，泌乳期Pten基因表達(dá)量顯著低于干乳期。與泌乳期低乳品質(zhì)奶牛乳腺相比，泌乳期高乳品質(zhì)的奶牛乳腺組織中Pten mRNA和蛋白表達(dá)水平分別降低了30%和40%；Pten基因過表達(dá)可抑制奶牛乳腺上皮細(xì)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），使奶牛乳腺上皮細(xì)胞中MAPK、CyclinD1、AKT、mTOR、S6K1、STAT5、SREBP1、PPAR？、PRLR、GLUT1的表達(dá)下調(diào)（P<0.05），并上調(diào)4EBP1的表達(dá)水平（P<0.05）；Pten基因抑制實(shí)驗(yàn)表現(xiàn)出相反的結(jié)果；而Pten基因的過表達(dá)和抑制對ELF5的表達(dá)均無顯著影響（P>0.05）。催乳素的添加能促進(jìn)細(xì)胞分泌？-酪蛋白、甘油三酯和乳糖（P<0.05），并下調(diào)Pten基因表達(dá)量（P<0.05）；葡萄糖的添加顯著增加了？-酪蛋白和乳糖分泌量（P<0.05），但甘油三酯含量無顯著變化（P>0.05），同時Pten基因的表達(dá)量無明顯改變（P>0.05）。綜上所述，Pten基因參與調(diào)節(jié)奶牛乳腺上皮細(xì)胞泌乳的過程，負(fù)向調(diào)節(jié)細(xì)胞的活力、增殖能力和細(xì)胞周期，并能抑制奶牛乳腺上皮細(xì)胞分泌？-酪蛋白、甘油三酯和乳糖；這種調(diào)節(jié)作用是通過Pten基因靶向調(diào)節(jié)PI3K-AKT信號通路，進(jìn)而調(diào)節(jié)其他泌乳相關(guān)信號通路基因的表達(dá)而實(shí)現(xiàn)的；同時發(fā)現(xiàn)Pten基因的表達(dá)受催乳素的負(fù)調(diào)節(jié)，但葡萄糖對Pten基因的表達(dá)水平無顯著影響。

關(guān)鍵詞：奶牛乳腺 發(fā)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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