干扰PPARγ基因对绵羊滋养层细胞增殖、凋亡、迁移和脂质积累的影响

doi:10.11843/j.issn.0366-6964.2024.06.014

摘要/Abstract

摘要：

旨在通过干扰PPARγ基因的表达，探究PPARγ对绵羊滋养层细胞增殖活性、细胞凋亡、细胞迁移率以及脂质累积的影响，为深入研究PPARγ在绵羊滋养层细胞中的分子机制提供基础依据。本研究设计并合成干扰PPARγ基因的siRNA干扰片段，通过Western Blot和RT-qPCR筛选干扰效率最高的siRNA，并转染至分离培养的绵羊滋养层细胞中。通过BODIPY染色检测干扰PPARγ基因后滋养层细胞中脂滴的聚积，比色法检测细胞中甘油三酯的含量，并利用RT-qPCR检测脂代谢相关基因的转录水平。采用CCK-8法、划痕试验和流式细胞法，分别探究干扰PPARγ基因对滋养层细胞增殖活性、细胞迁移率及细胞凋亡的影响。筛选得到了干扰效率最高的siPPARγ-1片段用于转染滋养层细胞，在干扰滋养层细胞PPARγ基因表达后，细胞中脂滴聚积能力下降，甘油三酯含量降低(P＜0.01)，脂代谢相关基因CD36、FABP4和PLIN2的转录水平也受到抑制(P＜0.01)。同时，滋养层细胞的增殖活力也显著降低(P＜0.05)，细胞迁移率减少(P＜0.01)，而细胞凋亡率显著上升(P＜0.01)。本研究表明PPARγ基因在调控滋养层细胞功能方面起重要作用，可对其具体分子机制进行深入研究，以期用于繁殖育种实践中。

关键词: 绵羊, 滋养层细胞, PPARγ, 干扰

Abstract:

The aim of this study was to explore the effects of PPARγ on the cell viability, apoptosis, cell migration rate and lipid accumulation of sheep trophoblast cells by interfering with the expression of PPARγ gene, so as to provide a basis for further study of the molecular mechanism of PPARγ in trophoblast cells.To design and synthesize siRNA interference fragment of PPARγ gene, the siRNA with the highest interference efficiency was screened by Western blot and RT-qPCR. Then, the siRNA was transfected into the sheep trophoblast cells. After interfering with the PPARγ gene, the accumulation of lipid droplets in trophoblast cells was measured by BODIPY staining, the amount of triglycerides by colorimetric method, and the transcript levels of genes involved in lipid metabolism was detected using RT-qPCR (real-time quantitative PCR). CCK-8 assay, wound healing test and flow cytometry were used to detect the effects of PPARγ interference on cell viability, migration rate and apoptosis of trophoblast cells. siPPARγ-1 fragment with the highest interference efficiency was screened and used to transfect trophoblast cells to interfere with the expression of PPARγ gene. Interference with PPARγ gene expression in trophoblast cells reduced the accumulation capacity of lipid droplets and cellular triglyceride content (P < 0.01), and the transcription levels of related genes CD36, FABP4 and PLIN2 were also inhibited (P < 0.01). At the same time, interference with PPARγ gene expression significantly reduced cell viability (P < 0.01) and migration rate (P < 0.05), while the cell apoptosis rate was increased significantly (P < 0.01).This study showed that PPARγ gene plays an important role in regulating the function of trophoblast cells and its specific molecular mechanism can be studied in depth with a view to using it in reproductive breeding practices.

Key words: sheep, trophoblast cells, PPARγ, interference

中图分类号:

S826.3

刘畅, 郝科兴, 陈岩, 曾维斌, 喻恒彬, 陈磊, 王静, 胡广东. 干扰PPARγ基因对绵羊滋养层细胞增殖、凋亡、迁移和脂质积累的影响[J]. 畜牧兽医学报, 2024, 55(6): 2421-2430.

Chang LIU, Kexing HAO, Yan CHEN, Weibin ZENG, Hengbin YU, Lei CHEN, Jing WANG, Guangdong HU. Effects of Interference with PPARγ Gene on Proliferation, Apoptosis, Migration and Lipid Accumulation of Trophoblast Cells in Sheep[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(6): 2421-2430.

图/表 7

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基因 Gene	GenBank号 GenBank No.	序列(5′→3′) Sequence	产物长度/bp Product length
CD36	XM_027968558.1	F：TGCCAGTTGGAGACATGCTCAT	154
		R：ACTGCCACTTCGTCTGGATTCT
FABP4	NM_001114667.1	F：ATGAAGGTGCTCTGGTACAAGT	135
		R：ATGCTCTCTCGTAAACTCTGGT
PLIN2	NM_001104932.1	F：CGCAATGCTGCCTCCTTT	131
		R：AGCCAGTTGAGGGGTGTGTT
PPARγ	KF727439.1	F：CTCATAATGCCATCAGGTTCG	102
		R：CAGCAGACTCTGGGTTCAGTTG

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