PPP5C基因调控牛脂肪细胞增殖、分化的功能研究

doi:10.11843/j.issn.0366-6964.2024.10.013

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (10): 4391-4402.doi: 10.11843/j.issn.0366-6964.2024.10.013

PPP5C基因调控牛脂肪细胞增殖、分化的功能研究

冯兰¹(), 冯雪¹, 马玉林¹, 张令锴¹, 马燕芬¹, 魏大为¹, 李芬², 张路培³, 杨润军⁴, 马云¹^,*(), 蔡蓓¹^,*()

1. 宁夏大学动物科技学院, 宁夏反刍动物分子细胞育种重点实验室, 银川 750021
2. 宁夏大学食品科学与工程学院, 银川 750021
3. 中国农业科学院北京畜牧兽医研究所, 北京 100193
4. 吉林大学动物科学学院, 长春 130000

收稿日期:2024-03-25 出版日期:2024-10-23 发布日期:2024-11-04
通讯作者: 马云,蔡蓓 E-mail:15202683541@163.com;mayun@nxu.edu.cn;caibei1115@163.com
作者简介:冯兰(1990-), 女, 宁夏吴忠人, 硕士生, 主要从事肉牛遗传育种研究, E-mail: 15202683541@163.com
基金资助:
国家自然科学基金项目(U22A20506);国家自然科学基金项目(32072720);宁夏回族自治区重点研发计划项目(23BCF01006);宁夏回族自治区重点研发计划项目(2021BEF01002);自治区科技创新领军人才培养项目(2020GKLRLX02)

Study on the Function of PPP5C Gene in Regulating the Proliferation and Differentiation of Bovine Adipocytes

Lan FENG¹(), Xue FENG¹, Yulin MA¹, Lingkai ZHANG¹, Yanfen MA¹, Dawei WEI¹, Fen LI², Lupei ZHANG³, Runjun YANG⁴, Yun MA¹^,*(), Bei CAI¹^,*()

1. Key Laboratory of Ruminants Molecular Cell Breeding, College of Animal Science and Technology, Ningxia University, Yinchuan 750021, China
2. School of Food Science and Engineering, Ningxia University, Yinchuan 750021, China
3. Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China
4. College of Animal Science, Jilin University, Changchun 130000, China

Received:2024-03-25 Online:2024-10-23 Published:2024-11-04
Contact: Yun MA, Bei CAI E-mail:15202683541@163.com;mayun@nxu.edu.cn;caibei1115@163.com

摘要/Abstract

摘要：

旨在探究PPP5C基因在牛脂肪细胞增殖、分化过程中的作用。本研究以固原黄牛为研究对象，在牛前体脂肪细胞中进行PPP5C基因功能缺失和功能获得性试验，利用qRT-PCR、EdU、CCK-8和流式细胞术检测候选标志基因的mRNA表达水平和细胞增殖活力，甘油三酯(TG)检测、油红O、BODIPY和Nile red染色结果为脂肪细胞分化过程中脂滴蓄积情况提供表型数据支撑。结果表明，相比对照组，干扰PPP5C基因显著上调增殖标志基因(CDK1、CDK2和PCNA)mRNA表达量(P < 0.01)，细胞活力显著上升(P < 0.01)，促进牛前体脂肪细胞从S期向G2期的转变(P < 0.01)。研究结果也提示，PPP5C可能也在牛前体脂肪分化过程中发挥负调控作用。主要表现在干扰PPP5C基因后，显著上调成脂标志基因(PPARγ、C/EBPα和FABP4)的mRNA表达(P < 0.01)，脂肪细胞中TG含量显著增多(P < 0.01)。同时，油红O、BODIPY和Nilered染色的细胞表型数据结果提示干扰PPP5C会促进牛前体脂肪细胞分化，脂滴蓄积增多。而PPP5C基因的功能获得性试验结果恰好与之相反。综上，干扰PPP5C基因后促进牛前体脂肪细胞增殖和分化，过表达PPP5C基因后抑制牛前体脂肪细胞增殖和分化，提示PPP5C基因可能在牛前体脂肪细胞增殖和分化过程中作为负调控因子发挥作用。本研究为进一步探究PPP5C基因调控牛脂肪沉积分子机制提供基础数据。

关键词: PPP5C, 牛, 前体脂肪细胞, 分化, 增殖, 流式细胞术

Abstract:

The aim of this study was to investigate the role of PPP5C gene in the proliferation and differentiation of bovine adipocytes. Loss-of-function and gain-of-function experiments of PPP5C gene were performed in precursor adipocytes of Guyuan cattle. mRNA expression levels of marker genes and cell proliferation viability were detected using qRT-PCR, EdU, CCK-8 and flow cytometry. The results of TG assay, Oil Red O, BODIPY and Nile red staining provided phenotypic data support of lipid droplets accumulation during adipocytes differentiation. The results showed that, compared with the control group, interfering with the PPP5C gene significantly up-regulated the mRNA expression of proliferation marker genes (CDK1, CDK2, and PCNA) (P < 0.01), and cell viability was significantly increased (P < 0.01), which facilitated the transition of bovine precursor adipocytes from the S-phase to the G2-phase (P < 0.01). The results also suggested that PPP5C might also play a negative regulatory role in bovine precursor adipose differentiation. This was mainly manifested in the significant up-regulation of mRNA expression of lipogenic marker genes (PPARγ, C/EBPα, and FABP4) (P < 0.01) and the significant increase of TG content in adipocytes (P < 0.01) after interfering with the PPP5C gene. Meanwhile, the results of cellular phenotypic data of Oil Red O, BODIPY and Nilered staining suggested that interfering with PPP5C promoted bovine precursor adipocyte differentiation and increased lipid droplet accumulation. The results of functional acquisition experiments with the PPP5C gene were just the opposite. In summary, interfering with the PPP5C gene promotes bovine precursor adipocyte proliferation and differentiation, and overexpression of the PPP5C gene inhibits bovine precursor adipocyte proliferation and differentiation, suggesting that the PPP5C gene may play a role as a negative regulator in bovine precursor adipocyte proliferation and differentiation. This study provides basic data for further investigation of the molecular mechanism of PPP5C gene in regulating bovine fat deposition.

Key words: PPP5C, bovine, precursor adipocytes, differentiation, proliferation, flow cytometry

中图分类号:

S823.2

冯兰, 冯雪, 马玉林, 张令锴, 马燕芬, 魏大为, 李芬, 张路培, 杨润军, 马云, 蔡蓓. PPP5C基因调控牛脂肪细胞增殖、分化的功能研究[J]. 畜牧兽医学报, 2024, 55(10): 4391-4402.

Lan FENG, Xue FENG, Yulin MA, Lingkai ZHANG, Yanfen MA, Dawei WEI, Fen LI, Lupei ZHANG, Runjun YANG, Yun MA, Bei CAI. Study on the Function of PPP5C Gene in Regulating the Proliferation and Differentiation of Bovine Adipocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(10): 4391-4402.

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名称 Name	上游引物序列(5′→3′) Upstream primer sequence	下游引物序列(5′→3′) Downstream primer sequence
Si-PPP5C-425	GGUGAAGGUGAAGCCACAUTT	AUGUGGCUUCACCUUCACCTT
Si-PPP5C-610	GCAGGGUGACAAUCACCUUTT	AAGGUGAUUGUCACCCUGCTT
Si-PPP5C-1081	CCCAGUGCAUCAAUGGCAATT	UUGCCAUUGAUGCACUGGGTT
对照Control	UUCUCCGAACGUGUCACGUTT	ACGUGACACGUUCGGAGAATT

基因 Gene	上游引物序列(5′→3′) Upstream primer sequence	下游引物序列(5′→3′) Downstream primer sequence
CDK1	GAAGGGGTTCCTAGTACTGC	ATGAACTGACCAGGAGGG
CDK2	ATGAACTGACCAGGAGGG	GCCAGGAGTTACTTCTATGC
PCNA	GAACCTCACCAGCATGTCCA	TACTAGTGCCAACGTGTCCG
PPARγ	AGGATGGGGTCCTCATATCC	GTCAGCTCTTGGGAACGGAA
C/EBPα	TGGACAAGAACAGCAACGAG	TTGTCACTGGTCAGCTCCAG
FABP4	AAGTCAAGAGCATCGTAA	CCAGCACCATCTTATCAT
GAPDH	CCAACGTGTCTGTTGTGGAT	CTGCTTCACCACCTTCTTGA

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PPP5C基因调控牛脂肪细胞增殖、分化的功能研究

Study on the Function of PPP5C Gene in Regulating the Proliferation and Differentiation of Bovine Adipocytes

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