沉默SCAP基因对奶牛乳腺上皮细胞脂滴的影响

doi:10.11843/j.issn.0366-6964.2019.03.005

摘要/Abstract

摘要：

旨在探究沉默固醇调节元件结合蛋白裂解激活蛋白（SREBP cleavage-activating protein，SCAP）对奶牛乳腺上皮细胞脂滴的影响，本研究构建了SCAP短发夹RNA慢病毒载体，包装感染奶牛乳腺上皮细胞，通过在培养基中添加嘌呤霉素筛选获得SCAP基因沉默稳转细胞株，采用Real-time PCR和Western blot观察基因沉默细胞株中SCAP基因和蛋白的表达，尼罗河红和油红O染色脂滴检测SCAP基因沉默及SCAP质粒瞬时转染对细胞脂滴形成的影响。结果表明，本研究成功构建了奶牛SCAP基因沉默慢病毒重组载体，感染细胞后经过5 mg·L^-1嘌呤霉素筛选得到SCAP基因沉默稳转细胞株。基因检测发现，各个沉默细胞株的SCAP基因表达显著降低（P<0.05或P<0.01）。与对照组相比，RNAi-SCAP3细胞中的SCAP蛋白表达降低为对照组的0.49倍（P<0.05），脂代谢基因SCD和FAS表达也显著下降（P<0.05）。脂滴染色发现，RNAi-SCAP3沉默细胞株的脂滴含量显著低于对照组细胞（P<0.01），脂滴直径≤ 2.0 μm的小脂滴个数增多，而直径≥ 2.5 μm的大脂滴个数减少；将SCAP真核表达载体瞬时转染RNAi-SCAP3细胞株后发现，SCAP基因的过表达显著减少了细胞中小脂滴比例而增加了大脂滴比例（P<0.05）。本研究成功构建了SCAP基因沉默稳转细胞株，发现SCAP的表达影响了细胞脂滴直径的大小。

Abstract:

The aim of this study was to investigate the effect of silencing SCAP gene on formation of lipid droplet in dairy mammary epithelial cells. SCAP short hairpin RNA lentivirus vector was constructed, the bovine mammary epithelial cells were packed and infected. SCAP gene silencing cell lines were selected by applying puromycin in culture medium. Effects of SCAP gene silencing on the expression of SCAP gene and protein were detected by Real-time PCR and Western blot, moreover, the effects of SCAP gene silencing and transient transfection of SCAP plasmid on lipid droplet formation were detected using Nile red and Oil red O staining. The results showed that lentivirus recombinant vector with SCAP gene silencing was constructed successfully. SCAP gene silencing stable cell line was obtained via screening of 5 mg·L^-1 puromycin. The expression of SCAP gene in all silent cell lines were significantly reduced(P<0.05 or P<0.01). Compared with control group, the expression of SCAP protein in RNAi-SCAP3 cell lines were decreased by 0.49 fold (P<0.05), the expression of SCD and FAS genes were significantly decreased (P<0.05) in RNAi-SCAP3 cells. Lipid droplet content in RNAi-SCAP3 cell lines was significantly lower than that in control cells (P<0.01). The number of small lipid droplets with size ≤ 2.0 μm were increased, while the number of large lipid droplets with size ≥ 2.5 μm were decreased. After transient transfection of SCAP eukaryotic expression vector into RNAi-SCAP3 gene silencing cell lines, overexpression of SCAP significantly decreased the percentage of small lipid droplets and increased the percentage of large lipid droplets (P<0.05). In this study, SCAP gene silencing cell lines were successfully constructed, and the size of lipid droplets were affected by the expression of SCAP.

中图分类号:

S823.2

邢智洋, 张梦璐, 张菡, 王月影, 韩立强, 高腾云, 杨国宇. 沉默SCAP基因对奶牛乳腺上皮细胞脂滴的影响[J]. 畜牧兽医学报, 2019, 50(3): 507-516.

XING Zhiyang, ZHANG Menglu, ZHANG Han, WANG Yueying, HAN Liqiang, GAO Tengyun, YANG Guoyu. Effect of Silencing SCAP Gene on Lipid Droplets in Dairy Mammary Epithelial Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(3): 507-516.

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https://www.xmsyxb.com/CN/Y2019/V50/I3/507

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