LncFAM200B对牦牛肌内前体脂肪细胞脂质沉积的影响

doi:10.3864/j.issn.0578-1752.2022.13.014

摘要/Abstract

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。

关键词: 牦牛, lncFAM200B, 肌内前体脂肪细胞, 超表达, 干扰

Abstract:

【Objective】The aim of this study was to analyze the effects of lncFAM200B in the lipid deposition in intramuscular preadipocytes of yak, which laid a foundation for further mechanism research. 【Method】 The longissimus dorsi muscle tissue of yak was collected and used to separate intramuscular preadipocytes. The adenovirus mediated overexpression technology was used to realize the overexpression of lncFAM200B, and the siRNA interference technology was used to analyze the function of lncFAM200B. The mRNA expression level of fat differentiation marker (PPARγ, C/EBPα and AP2) and the potential target (SIRT1 and PTEN) genes of lncFAM200B were detected via real-time fluorescent quantitative PCR (RT-qPCR). Oil red O staining, triacylglycerol (TAG) determination and CCK-8 determination methods were used to detect intracellular lipid droplet deposition and preadipocyte proliferation.【Result】The overexpression of lncFAM200B not only significantly increased the fat differentiation genes (C/EBPα and AP2, P<0.05) expression level, but also increased the lipid droplets deposition with large lipid droplets in the cells during induced differentiation. Conversely, lncFAM200B interference reduced the expression of PPARγ, C/EBPα and AP2 (P<0.05), and lipid droplet deposition. Furthermore, the triacylglycerol content after lncFAM200B overexpression 4 days was significantly higher than that of the control group (P<0.05), but lower after siRNA interference 6 days. Moreover, the SIRT1 levels increased with time was first decreased and then increased trend, and the PTEN had the opposite trend during lncFAM200B overexpression, and the opposite results of the two genes obtained during lncFAM200B interference. In addition, the results of CCK-8 experiment showed that there was a significant difference in cell proliferation activity both in overexpression or interference group after 72 hours. 【Conclusion】lncFAM200B could regulate yak adipose differentiation by influencing the expression of fat differentiation marker (C/EBPα and AP2), and further affect the triacylglycerol content and the lipid droplet deposition, but the detail mechanism need to be further research efforts.

Key words: yak, lncFAM200B, intramuscular preadipocytes, overexpression, interference

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.

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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