LPIN1基因对水牛乳腺上皮细胞甘油三酯和脂肪酸合成的影响

doi:10.16431/j.cnki.1671-7236.2020.10.012

摘要/Abstract

摘要： 本试验旨在通过构建腺病毒pDC316-LPIN1-eGFP载体并包装出相应的脂素腺病毒颗粒（Ad-LPIN1），探索LPIN1基因对水牛乳腺上皮细胞甘油酯和脂肪酸合成的影响。将Ad-LPIN1腺病毒颗粒感染水牛乳腺上皮细胞，通过实时荧光定量PCR、免疫荧光染色（immunofluorescence，IF）检测LPIN1基因和lipin1蛋白的表达情况；通过油红O染色和甘油三酯测定甘油三酯；通过实时荧光定量PCR检测LIPN1基因对甘油三酯和脂肪酸合成相关基因表达的影响。本试验成功构建pDC316-LPIN1-eGFP载体并获得高质量的Ad-LPIN1腺病毒颗粒。Ad-LPIN1腺病毒颗粒感染水牛乳腺上皮细胞（MOI=50）后LPIN1基因mRNA相对上调>5 000倍，差异极显著（P<0.01），lipin1蛋白表达量增多且主要在细胞核中表达。油红O染色结果显示，过表达LPIN1基因后细胞中的脂滴明显减少。甘油三酯测定结果显示，过表达LPIN1基因后细胞中甘油三酯含量下降，差异极显著（P<0.01）。实时荧光定量PCR检测表明，LPIN1基因过表达会使过氧化物酶体增殖物激活受体γ（peroxisome proliferator activated receptor gama，PPARγ）、过氧化物酶体增殖物激活受体α（peroxisome proliferator activated receptor alpha，PPARα）、固醇调节元件结合蛋白1（sterol regulatory element binding transcription factor 1，SREBP1）基因表达量显著或极显著上调（P<0.05；P<0.01），同时使硬脂酰辅酶A去饱和酶（stearoyl-CoA desaturase，SCD）、脂肪酸合成酶（fatty acid synthase，FASN）、长链脂酰CoA合成酶（acyl-CoA synthetase long chain family member 1，ACSL1）、乙酰辅酶A羧化酶α（acetyl-CoA carboxylase alpha，ACACA）基因表达量极显著下降，分别下调至83.17%、51.30%、44.24%和41.18%（P<0.01）。以上结果表明，腺病毒高效介导LPIN1基因在水牛乳腺上皮细胞中表达，lipin1蛋白在细胞核中的表达量增加，lipin1蛋白作为转录共同激活因子下调ACACA、FASN、ACSL1和SCD基因的表达，从而抑制甘油三酯的合成。

关键词: 水牛; 乳腺上皮细胞; LPIN1基因; 甘油三酯; 脂肪酸

Abstract: The purpose of this study was to construct the adenovirus pDC316-LPIN1-eGFP vector and package the Ad-LPIN1 adenovirus particles,in order to explore the effect of LPIN1 gene on triglyceride and fatty acid synthesis in buffalo mammary epithelial cells.The expression of LPIN1 gene and lipin1 protein in buffalo mammary epithelial cell were detected by Real-time quantitative PCR and immunofluorescence (IF) after Ad-LPIN1 infected.The triglyceride was detected by oil red O staining and triglyceride measurement and the genes expression of triglyceride synthesis and fatty acid synthesis after overexpressing LPIN1 were detected by Real-time quantitative PCR.The results showed that high-quality Ad-LPIN1 adenovirus particles were packaged with the pDC316-LPIN1-eGFP vector.Ad-LPIN1 adenovirus particles infected the buffalo mammary epithelial cell (MOI=50),LPIN1 mRNA was relatively up-regulated more than 5 000-fold,which was extremely significant difference (P<0.01).lipin1 protein expression was up-regulated and mainly expressed in the nucleus.Oil red O staining results showed that lipid droplets in cells were significantly reduced after overexpressing LPIN1 gene.The results of triglyceride measurement showed that the triglyceride content in cells decreased after overexpressing LPIN1 gene,and the difference was extremely significant (P<0.01).The mRNA of peroxisome proliferator activated receptor gama (PPARγ),peroxisome proliferator activated receptor alpha (PPARα) and sterol regulatory element binding transcription factor 1 (SREBP1) after overexpressing LPIN1 gene were significantly or extremely significant increased (P<0.05;P<0.01).And the mRNA of stearoyl-CoA desaturase (SCD),fatty acid synthase (FASN),acyl-CoA synthetase long chain family member 1 (ACSL1) and acetyl-CoA carboxylase alpha (ACACA) were decreased to 83.17%,51.30%,44.24%,41.18% (P<0.01),respectively.The above-mentioned results showed that adenovirus efficiently mediated the expression of LPIN1 gene in buffalo mammary epithelial cell,and increased the expression of lipin1 protein in the nucleus.lipin1 protein,as a common transcription activation factor,inhibited the synthesis of triglycerides by down-regulating mRNA of ACACA,FASN,ACSL1 and SCD genes.

Key words: buffalo; mammary epithelial cell; LPIN1 gene; triglyceride; fatty acid

中图分类号:

S823.8⁺3

段安琴, 陆杏蓉, 马小娅, 梁莎莎, 邓廷贤. LPIN1基因对水牛乳腺上皮细胞甘油三酯和脂肪酸合成的影响[J]. 中国畜牧兽医, 2020, 47(10): 3140-3148.

DUAN Anqin, LU Xingrong, MA Xiaoya, LIANG Shasha, DENG Tingxian. Effect of LPIN1 Gene on Triglyceride and Fatty Acid Synthesis in Buffalo Mammary Epithelial Cell[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(10): 3140-3148.

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