中国农业科学 ›› 2022, Vol. 55 ›› Issue (13): 2654-2666.doi: 10.3864/j.issn.0578-1752.2022.13.014
冉宏标(),赵丽玲,王会(
),柴志欣,王吉坤,王嘉博,武志娟,钟金城(
)
收稿日期:
2021-05-12
接受日期:
2021-12-16
出版日期:
2022-07-01
发布日期:
2022-07-08
联系方式:
冉宏标,Tel:17761228950;E-mail: haluxi_7@163.com。
基金资助:
RAN HongBiao(),ZHAO LiLing,WANG Hui(
),CHAI ZhiXin,WANG JiKun,WANG JiaBo,WU ZhiJuan,ZHONG JinCheng(
)
Received:
2021-05-12
Accepted:
2021-12-16
Published:
2022-07-01
Online:
2022-07-08
摘要:
【目的】拟通过腺病毒介导的超表达及siRNA干扰试验,分析lncFAM200B对牦牛肌内脂肪细胞脂质沉积的影响,为解析lncFAM200B对牦牛肌内脂肪细胞脂质沉积的调控机制奠定基础。【方法】采集牦牛背最长肌组织,采用酶消化法和差速贴壁法分离牦牛肌内前体脂肪细胞;构建包装lncFAM200B超表达腺病毒,设计合成其siRNA干扰序列;通过实时荧光定量PCR(RT-qPCR)分析lncFAM200B超表达与干扰后脂肪分化标志基因PPARγ、C/EBPα、AP2,lncFAM200B潜在靶基因SIRT1、PTEN的表达水平;采用油红O染色、甘油三酯(TAG)测定、CCK-8检测等方法检测细胞内脂滴沉积情况、甘油三酯含量变化及细胞增殖情况。【结果】从牦牛背最长肌成功分离获得肌内前体脂肪细胞;lncFAM200B超表达后,脂肪分化标志基因C/EBPα、AP2表达量显著升高(P<0.05),随着诱导分化时间的增加,lncFAM200B潜在靶基因SIRT1表达量呈先降低后升高的趋势,PTEN表达量呈现先增高后降低趋势;细胞脂滴沉积显著增加(P<0.05),且胞内形成较大脂滴;超表达4 d后,细胞内甘油三酯含量显著增加(P<0.05)。干扰lncFAM200B可显著降低脂肪分化标志基因PPARγ、C/EBPα、AP2的表达水平(P<0.05),降低细胞脂肪沉积,且诱导分化第6天细胞内甘油三酯含量显著降低(P<0.05);干扰lncFAM200B后其潜在靶基因SIRT1呈现先升高后降低的表达趋势,PTEN则相反。CCK-8增殖实验表明,lncFAM200B超表达72 h后,细胞增殖效率显著降低(P<0.05),而干扰lncFAM200B后72 h后细胞增殖效率显著增强(P<0.05)。【结论】lncFAM200B可能通过影响脂肪分化标志基因C/EBPα和AP2,脂肪合成相关基因SIRT1和PTEN的表达从而影响牦牛肌内脂肪沉积,但其具体机制还需要进一步研究。
冉宏标,赵丽玲,王会,柴志欣,王吉坤,王嘉博,武志娟,钟金城. LncFAM200B对牦牛肌内前体脂肪细胞脂质沉积的影响[J]. 中国农业科学, 2022, 55(13): 2654-2666.
RAN HongBiao,ZHAO LiLing,WANG Hui,CHAI ZhiXin,WANG JiKun,WANG JiaBo,WU ZhiJuan,ZHONG JinCheng. Effects of lncFAM200B on the Lipid Deposition in Intramuscular Preadipocytes of Yak[J]. Scientia Agricultura Sinica, 2022, 55(13): 2654-2666.
表1
RT-qPCR引物序列"
基因名称 Gene name | 登录号 Login ID | 引物序列 (5′-3′) Primer sequence (5′-3′) | 产物长度 Product size (bp) |
---|---|---|---|
lncFAM200B | F:GCTTCCCATCAGAAAGTATCAGG R:TTGTGTTGGTAGCTTGACTACG | 141 | |
GAPDH | NM_001034034.2 | F:CCACGAGAAGTATAACAACACC R:GTCATAAGTCCCTCCACGAT | 120 |
SIRT1 | XM_024986766.1 | F:TGGGGTTTCTGTTTCTTGTGG R:CTTGAGGATCTGGAAGGTCTGG | 98 |
PTEN | NM_001319898.1 | F:GGAAAGGGACGAACTGGTGTAA R:TGTCTCTGGTCCTTACTTCCCC | 109 |
AP2 | XM_005897260 | F:GGAAAGTCAAGAGCATCGTAAAC R:CACCATCTTATCATCCACGAGTT | 110 |
C/EBPα | NM_176784.2 | F:GTGGACAAGAACAGCAACGAGTA R:GCGGTCATTGTCACTGGTCAG | 138 |
PPARγ | NM_181024.2 | F:CGTGGACCTTTCTATGATGGA R:GCTCTTGGGAACGGAATG | 117 |
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