目的:肌肉乙酰胆碱受体具有五个α亚基(α,β,δ,ε,或γ),胆碱能受体烟碱γ亚基(CHRNG)是γ亚基。它也可能在生物过程中发挥重要作用,包括细胞分化,增长,和生存,而CHRNG的作用尚未在文献中研究过。因此,本研究旨在阐明CHRNG对牛前脂肪细胞增殖和分化的影响。
方法:我们构建了一个CHRNG过表达腺病毒载体,并成功地在牛前脂肪细胞上过表达。用Edu法检测CHRNG对牛前脂肪细胞增殖的影响,细胞计数试剂盒-8(CCK-8),实时荧光定量聚合酶链反应(RT-qPCR),Westernblot等技术。我们还表演了油红O,RT-qPCR,Westernblot探讨其对前脂肪细胞分化的影响。
结果:Edu增殖实验结果表明,过表达组的EDU阳性细胞数量明显减少。CCK-8实验发现过表达组细胞的光密度值低于对照组,增殖细胞核抗原(PCNA)的mRNA水平,细胞周期蛋白A2(CCNA2),细胞周期蛋白B1(CCNB1),细胞周期蛋白D2(CCND2)在CHRNG基因过表达后显著降低,细胞周期蛋白依赖性激酶抑制剂1A(CDKN1A)的mRNA水平显着增加,和PCNA的蛋白质水平,CCNB1、CCND2显著降低。CHRNG的过表达抑制了牛前脂肪细胞的分化。油红O和甘油三酯测定结果表明,过表达组脂滴积累的大小和速度均明显降低。过氧化物酶体增殖物激活受体γ(PPARγ)的mRNA和蛋白水平,CCAAT增强子结合蛋白α(CEBPα),脂肪酸结合蛋白4(FABP4),脂肪酸合成酶(FASN)显著降低。
结论:CHRNG在牛前脂肪细胞中的过表达抑制了牛前脂肪细胞的增殖和分化。
OBJECTIVE: Muscle acetylcholine receptors have five alpha subunits (α, β, δ, ε, or γ), and cholinergic receptor nicotinic gamma subunit (
CHRNG) is the γ subunit. It may also play an essential role in biological processes, including cell differentiation, growth, and survival, while the role of
CHRNG has not been studied in the literature. Therefore, the purpose of this study is to clarify the effect of CHRNG on the proliferation and differentiation of bovine preadipocytes.
METHODS: We constructed a
CHRNG overexpression adenovirus vector and successfully overexpressed it on bovine preadipocytes. The effects of
CHRNG on bovine preadipocyte proliferation were detected by Edu assay, cell counting Kit-8 (CCK-8), real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), Western blot and other techniques. We also performed oil red O, RT-qPCR, Western blot to explore its effect on the differentiation of preadipocytes.
RESULTS: The results of Edu proliferation experiments showed that the number of EDU-positive cells in the overexpression group was significantly less. CCK-8 experiments found that the optical density values of the cells in the overexpression group were lower than those of the control group, the mRNA levels of proliferating cell nuclear antigen (PCNA), cyclin A2 (CCNA2), cyclin B1 (CCNB1), cyclin D2 (CCND2) decreased significantly after CHRNG gene overexpression, the mRNA levels of cyclin dependent kinase inhibitor 1A (CDKN1A) increased significantly, and the protein levels of PCNA, CCNB1, CCND2 decreased significantly. Overexpression of
CHRNG inhibited the differentiation of bovine preadipocytes. The results of oil red O and triglyceride determination showed that the size and speed of lipid droplets accumulation in the overexpression group were significantly lower. The mRNA and protein levels of peroxisome proliferator activated receptor gamma (PPARγ), CCAAT enhancer binding protein alpha (CEBPα), fatty acid binding protein 4 (FABP4), fatty acid synthase (FASN) decreased significantly.
CONCLUSIONS: Overexpression of CHRNG in bovine preadipocytes inhibits the proliferation and differentiation of bovine preadipocytes.