Article(id=1307013785093435898, tenantId=1146029695717560320, journalId=1306274286668972124, issueId=1307013423896753139, articleNumber=null, orderNo=null, doi=10.11843/j.issn.0366-6964.2026.03.017, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1750953600000, receivedDateStr=2025-06-27, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1789545969507, onlineDateStr=2026-09-16, pubDate=1774195200000, pubDateStr=2026-03-23, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1789545969507, onlineIssueDateStr=2026-09-16, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1789545969507, creator=13701087609, updateTime=1789545969507, updator=13701087609, issue=Issue{id=1307013423896753139, tenantId=1146029695717560320, journalId=1306274286668972124, year='2026', volume='57', issue='3', pageStart='1177', pageEnd='1776', issueExtLink='null', onlineDate='null', pubDate='1774195200000', pubDateStr='2026-03-23', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=1, specialIssue=null, createTime=1789545883392, creator='13701087609', updateTime=1789609930841, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1307282058464743901, tenantId=1146029695717560320, journalId=1306274286668972124, issueId=1307013423896753139, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1307282058464743902, tenantId=1146029695717560320, journalId=1306274286668972124, issueId=1307013423896753139, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=1362, endPage=1372, ext={EN=ArticleExt(id=1307013785307345403, articleId=1307013785093435898, tenantId=1146029695717560320, journalId=1306274286668972124, language=EN, title=6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase 3 (PFKFB3) Promotes Adipogenic Differentiation of Porcine Intramuscular Preadipocytes and Screening of Its Interacting Proteins, columnId=1307007413870158054, journalTitle=Acta Veterinaria et Zootechnica Sinica, columnName=ANIMAL GENETICS AND BREEDING, runingTitle=null, highlight=null, articleAbstract=

This study aimed to investigate the regulatory role of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) in the proliferation and adipogenic differentiation of porcine intramuscular preadipocytes, screen its interacting proteins and provide clues for understanding their regulatory mechanisms. PFKFB3 was overexpressed or interfered in porcine intramuscular preadipocytes. qRT-PCR was used to detect changes in the expression of proliferation marker genes. CCK-8 and EdU assays were employed to measure changes in the number of viable cells and cells in the proliferation phase, respectively. After inducing adipogenic differentiation of porcine intramuscular preadipocytes, qRT-PCR and Oil Red O staining were used to assess its regulatory effect on adipogenic differentiation. Co-immunoprecipitation combined with liquid chromatography-tandem mass spectrometry was utilized to identify and screen proteins interacting with PFKFB3. The results showed that overexpression of PFKFB3 significantly increased the number of EdU-positive cells (P<0.01) and cell viability (P<0.01), and significantly upregulated the expression of proliferation-related genes CDK1, CCND1, PCNA, and KI67 (P<0.05). Conversely, interfering with PFKFB3 significantly inhibited cell proliferation (P<0.05). Regarding adipogenic differentiation, overexpression of PFKFB3 significantly increased the number of lipid droplets (P<0.01) and triglyceride content (P<0.01), and significantly upregulated the expression of key adipogenic genes PPARγ, CEBPα, CEBPβ, and LPL (P<0.05). Interfering with PFKFB3 significantly inhibited adipogenic differentiation (P<0.05). Furthermore, Flag-tag immunoprecipitation combined with mass spectrometry analysis identified 17 specific interacting proteins of PFKFB3. GO analysis indicated that these proteins were primarily involved in processes such as ATP binding and RNA binding. STRING protein-protein interaction network prediction revealed interactions between PFKFB3 and proteins including CHORDC1, ALAS1, ASCC3, and KCNK2. In summary, PFKFB3 positively regulates the proliferation and adipogenic differentiation of porcine intramuscular preadipocytes. Potential interacting proteins of PFKFB3 were preliminarily screened, providing new clues for in-depth analysis of the molecular mechanisms underlying porcine intramuscular fat deposition and improvement of pork quality.

, authors=Meng LI, Jiayi CHEN, Mingxin WEN, Yuqi HAN, Shaojie QIAN, Zumei MING, Xiaohong GUO, Bugao LI, authorsList=Meng LI, Jiayi CHEN, Mingxin WEN, Yuqi HAN, Shaojie QIAN, Zumei MING, Xiaohong GUO, Bugao LI, authorCompany=null, correspAuthors=Bugao LI, authorNote=null, correspAuthorsNote=
LI Bugao, E-mail:
, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=null), CN=ArticleExt(id=1307013789094801931, articleId=1307013785093435898, tenantId=1146029695717560320, journalId=1306274286668972124, language=CN, title=6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶3(PFKFB3)促进猪肌内前体脂肪细胞成脂分化及其互作蛋白的筛选, columnId=1307007414092456168, journalTitle=畜牧兽医学报, columnName=遗传育种, runingTitle=null, highlight=null, articleAbstract=

旨在探究6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶3(6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3,PFKFB3)对猪肌内前体脂肪细胞增殖和成脂分化的调控作用,并筛选其互作蛋白,为解析其调控机制提供线索。本研究通过在猪肌内前体脂肪细胞中过表达/干扰PFKFB3,采用qRT-PCR检测增殖标志基因的表达变化,采用CCK-8、EdU试验检测活细胞及处于增殖期细胞数量的变化;诱导猪肌内前体脂肪细胞成脂分化后,通过qRT-PCR、油红O等技术,检测其对细胞成脂分化的调控作用;运用免疫共沉淀及液相色谱-质谱技术识别和筛选PFKFB3的互作蛋白。结果显示,过表达PFKFB3后EdU阳性细胞数量显著增加(P<0.01),细胞活力显著升高(P<0.01),且增殖相关基因CDK1、CCND1、PCNA、KI67表达显著上调(P<0.05);干扰PFKFB3则显著抑制细胞增殖(P<0.05)。成脂分化方面,过表达PFKFB3 可显著增加脂滴数量(P<0.01)和甘油三酯含量(P<0.01),并显著上调成脂关键基因PPARγ、CEBPα、CEBPβ、LPL等的表达(P<0.05);干扰PFKFB3显著抑制细胞的成脂分化(P<0.05)。此外,通过Flag标签免疫沉淀结合质谱分析共筛选到17个PFKFB3的特异性互作蛋白。GO分析表明这些蛋白主要参与ATP结合和RNA结合等过程;STRING蛋白互作网络预测显示PFKFB3与CHORDC1、ALAS1、ASCC3、KCNK2等蛋白存在相互作用。综上,PFKFB3可正向调控猪肌内前体脂肪细胞的增殖和成脂分化,并初步筛选出其潜在互作蛋白,为深入解析猪肌内脂肪沉积的分子机制及改善猪肉品质提供了新线索。

, authors=李萌, 陈佳仪, 文明昕, 韩雨琪, 钱少杰, 明祖梅, 郭晓红, 李步高, authorsList=李萌, 陈佳仪, 文明昕, 韩雨琪, 钱少杰, 明祖梅, 郭晓红, 李步高, authorCompany=null, correspAuthors=李步高, authorNote=

李萌,博士,主要从事动物遗传育种与繁殖的研究,E-mail:

李萌和陈佳仪为同等贡献作者

陈佳仪,硕士,主要从事动物遗传育种与繁殖的研究,E-mail:

, correspAuthorsNote=
李步高,主要从事动物遗传育种与繁殖的研究,E-mail:
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李萌和陈佳仪为同等贡献作者

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陈佳仪,硕士,主要从事动物遗传育种与繁殖的研究,E-mail:

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陈佳仪,硕士,主要从事动物遗传育种与繁殖的研究,E-mail:

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A. Adipogenic differentiation, day 2; B. Adipogenic differentiation, day 8

, figureFileSmall=DvZKYSz7+UVVNkHSQXxKxA==, figureFileBig=guHA13q/M/SOWwSan2B25w==, tableContent=null), ArticleFig(id=1307281541692941205, tenantId=1146029695717560320, journalId=1306274286668972124, articleId=1307013785093435898, language=CN, label=图1, caption=猪肌内前体脂肪细胞油红O染色结果

A.成脂分化2 d;B.成脂分化8 d

, figureFileSmall=DvZKYSz7+UVVNkHSQXxKxA==, figureFileBig=guHA13q/M/SOWwSan2B25w==, tableContent=null), ArticleFig(id=1307281541797798806, tenantId=1146029695717560320, journalId=1306274286668972124, articleId=1307013785093435898, language=EN, label=Fig.2, caption=Effect of overexpression of PFKFB3 on proliferation of porcine intramuscular preadipocytes

A. PFKFB3 overexpression efficiency; B. EdU staining assay, EdU-positive cells were red and nuclei were blue; C. Quantification of EdU staining results; D. Results of CCK-8 assay; E. Effects of overexpression of PFKFB3 on the expression of genes related to cell proliferation. *. P<0.05, **. P<0.01, the same as below

, figureFileSmall=8sPTVFIWj/mK59l6oL3byw==, figureFileBig=5ksRwyFEYHkUfDI/hSPQhQ==, tableContent=null), ArticleFig(id=1307281542045262743, tenantId=1146029695717560320, journalId=1306274286668972124, articleId=1307013785093435898, language=CN, label=图2, caption=过表达PFKFB3对猪肌内前体脂肪细胞增殖的影响

A. PFKFB3过表达效率;B.EdU试验,阳性细胞呈红色,细胞核呈蓝色;C. EdU染色结果量化;D. CCK-8试验结果;E.过表达PFKFB3对细胞增殖相关基因表达的影响。*.P<0.05,**.P<0.01,下同

, figureFileSmall=8sPTVFIWj/mK59l6oL3byw==, figureFileBig=5ksRwyFEYHkUfDI/hSPQhQ==, tableContent=null), ArticleFig(id=1307281543714595736, tenantId=1146029695717560320, journalId=1306274286668972124, articleId=1307013785093435898, language=EN, label=Fig.3, caption=Effect of interfering with PFKFB3 on the cell proliferation

A.PFKFB3 interference efficiency; B. EdU staining assay; C. Quantification of EdU staining results; D. Results of the CCK-8 assay; E. Effects of interfering with PFKFB3 on the expression of genes related to cell proliferation

, figureFileSmall=cunJvHl/O87/44Oh9AMXQg==, figureFileBig=xLmVBZGVzFO946vQWnsgBw==, tableContent=null), ArticleFig(id=1307281543798481817, tenantId=1146029695717560320, journalId=1306274286668972124, articleId=1307013785093435898, language=CN, label=图3, caption=干扰PFKFB3对细胞增殖的影响

A. PFKFB3干扰效率;B.EdU试验;C. EdU染色结果量化;D. CCK-8试验结果;E.干扰PFKFB3对细胞增殖相关基因的表达变化

, figureFileSmall=cunJvHl/O87/44Oh9AMXQg==, figureFileBig=xLmVBZGVzFO946vQWnsgBw==, tableContent=null), ArticleFig(id=1307281544142414746, tenantId=1146029695717560320, journalId=1306274286668972124, articleId=1307013785093435898, language=EN, label=Fig.4, caption=Effect of overexpression of PFKFB3 on porcine intramuscular preadipocytes differentiation

A, B. Oil red O staining test results; C. Effects of PFKFB3 overexpression on TG accumulation in porcine intramuscular preadipocytes; D. Effect of overexpression of PFKFB3 on genes expression related to lipogenic differentiation of cells

, figureFileSmall=u2xOaufBQLMC3QXjE4dl4g==, figureFileBig=NWUv9GSsQXF4iMKya3JUXQ==, tableContent=null), ArticleFig(id=1307281544222106523, tenantId=1146029695717560320, journalId=1306274286668972124, articleId=1307013785093435898, language=CN, label=图4, caption=过表达PFKFB3对猪肌内前体脂肪细胞分化的影响

A、B.油红O染色试验结果;C.过表达PFKFB3对猪肌内前体脂肪细胞中TG含量的影响;D.过表达PFKFB3对成脂分化相关基因表达的影响

, figureFileSmall=u2xOaufBQLMC3QXjE4dl4g==, figureFileBig=NWUv9GSsQXF4iMKya3JUXQ==, tableContent=null), ArticleFig(id=1307281544305992604, tenantId=1146029695717560320, journalId=1306274286668972124, articleId=1307013785093435898, language=EN, label=Fig.5, caption=Effect of interfering with PFKFB3 on porcine intramuscular preadipocytes differentiation

A, B. Oil red O staining test results; C. The effect of interfering with PFKFB3 on the TG content in porcine intramuscular preadipocytes; D. Effect of interfering with PFKFB3 on genes expression related to lipogenic differentiation of cells

, figureFileSmall=X+K+A93YbXGI2BlX5Ir6xg==, figureFileBig=ctirwldsaTqkQNqozGKUpQ==, tableContent=null), ArticleFig(id=1307281544389878685, tenantId=1146029695717560320, journalId=1306274286668972124, articleId=1307013785093435898, language=CN, label=图5, caption=干扰PFKFB3对猪肌内前体脂肪细胞分化的影响

A、B.油红O染色试验结果;C.干扰PFKFB3对猪肌内前体脂肪细胞中TG含量的影响;D.干扰PFKFB3对细胞成脂分化相关基因表达的影响

, figureFileSmall=X+K+A93YbXGI2BlX5Ir6xg==, figureFileBig=ctirwldsaTqkQNqozGKUpQ==, tableContent=null), ArticleFig(id=1307281544561845150, tenantId=1146029695717560320, journalId=1306274286668972124, articleId=1307013785093435898, language=EN, label=Fig.6, caption=Results of silver staining and mass spectrometry identification analysis of PFKFB3 Pull-down proteins

A. Silver staining results of PFKFB3 interacting proteins; B. Venn diagram of PFKFB3 interacting proteins; C. GO functional annotation analysis results of PFKFB3 interacting proteins; D. Proteins interaction network diagram based on PFKFB3 interacting proteins

, figureFileSmall=m6oUYLVUCMgMezYDPaAPWg==, figureFileBig=I0Xn2LEoEM4l1Vix06w96g==, tableContent=null), ArticleFig(id=1307281544637342623, tenantId=1146029695717560320, journalId=1306274286668972124, articleId=1307013785093435898, language=CN, label=图6, caption=PFKFB3 Pull-down蛋白的银染及质谱鉴定分析结果

A. PFKFB3互作蛋白银染结果;B. PFKFB3互作蛋白韦恩图;C. PFKFB3互作蛋白GO功能注释分析结果;D.基于PFKFB3互作蛋白绘制的蛋白相互作用网络关系图

, figureFileSmall=m6oUYLVUCMgMezYDPaAPWg==, figureFileBig=I0Xn2LEoEM4l1Vix06w96g==, tableContent=null), ArticleFig(id=1307281544708645792, tenantId=1146029695717560320, journalId=1306274286668972124, articleId=1307013785093435898, language=EN, label=Table 1, caption=

Primer sequences of qRT-PCR

, figureFileSmall=null, figureFileBig=null, tableContent=

基因

Gene

引物序列(5'→3')

Primer sequence

产物长度/bp

Product length

PFKFB3

F: ATGAGAGTCCGCAAGCAGTGT

R: TCTCTAGTAGTGTTGGTGGCATCA

110
CDK1

F: GCTTTTCAAAGCTGGCTCTGGG

R: TGGATGTGGTAGATCCCAGCTTA

104
CCND1

F: CTCCGTATCTTACTTCAAGTGCG

R: CTTCTCGGCAGTCAAGGGAA

250
KI67

F: ATCATTGACCGCTCCTTTAGGT

R: GCTCGCCTTGATGGTTCCT

104
PCNA

F: GCAGAGCATGGACTCGTCTC

R: TTGGACATGCTGGTGAGGTT

150
PPARγ

F: TGGGTGAAACTCTGGGAGATTC

R: AGAGGTCCACAGAGCTGATTCC

150
CEBPα

F: CAAGAACAGCAACGAGTACCG

R: GTCACTGGTCAACTCCAGCAC

124
CEBPβ

F: AAGAGTAAGACCAAGAAGACC

R: GCTCCAGGACCTTATGCT

139
FABP4

F: ATGTCTAGCAACAGTTTCGCC

R: TCGCTTCATAGCATCTAGCTTG

195
LPL

F: CGACTCTCTGTTGAATGAAG

R: TTGGCTCTGACCTTATTGATC

141
Perilipin-1

F: TCCAGCCAAGGAAGAGTCAG

R: GGAAGGTGTGTTGAGAGATGG

125
AdipoQ

F: AGGCTGGATGGCGGGTAT

R: ACACTGAATGCTGAACGGTAG

240
18S rRNA

F: ATAAACGATGCCGACTGGCGAT

R: CAATCTGTCAATCCTGTCCGTGT

245
), ArticleFig(id=1307281544771560353, tenantId=1146029695717560320, journalId=1306274286668972124, articleId=1307013785093435898, language=CN, label=表1, caption=

qRT-PCR引物信息

, figureFileSmall=null, figureFileBig=null, tableContent=

基因

Gene

引物序列(5'→3')

Primer sequence

产物长度/bp

Product length

PFKFB3

F: ATGAGAGTCCGCAAGCAGTGT

R: TCTCTAGTAGTGTTGGTGGCATCA

110
CDK1

F: GCTTTTCAAAGCTGGCTCTGGG

R: TGGATGTGGTAGATCCCAGCTTA

104
CCND1

F: CTCCGTATCTTACTTCAAGTGCG

R: CTTCTCGGCAGTCAAGGGAA

250
KI67

F: ATCATTGACCGCTCCTTTAGGT

R: GCTCGCCTTGATGGTTCCT

104
PCNA

F: GCAGAGCATGGACTCGTCTC

R: TTGGACATGCTGGTGAGGTT

150
PPARγ

F: TGGGTGAAACTCTGGGAGATTC

R: AGAGGTCCACAGAGCTGATTCC

150
CEBPα

F: CAAGAACAGCAACGAGTACCG

R: GTCACTGGTCAACTCCAGCAC

124
CEBPβ

F: AAGAGTAAGACCAAGAAGACC

R: GCTCCAGGACCTTATGCT

139
FABP4

F: ATGTCTAGCAACAGTTTCGCC

R: TCGCTTCATAGCATCTAGCTTG

195
LPL

F: CGACTCTCTGTTGAATGAAG

R: TTGGCTCTGACCTTATTGATC

141
Perilipin-1

F: TCCAGCCAAGGAAGAGTCAG

R: GGAAGGTGTGTTGAGAGATGG

125
AdipoQ

F: AGGCTGGATGGCGGGTAT

R: ACACTGAATGCTGAACGGTAG

240
18S rRNA

F: ATAAACGATGCCGACTGGCGAT

R: CAATCTGTCAATCCTGTCCGTGT

245
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6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶3(PFKFB3)促进猪肌内前体脂肪细胞成脂分化及其互作蛋白的筛选
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李萌 , 陈佳仪 , 文明昕 , 韩雨琪 , 钱少杰 , 明祖梅 , 郭晓红 , 李步高
畜牧兽医学报 | 遗传育种 2026,57(3): 1362-1372
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畜牧兽医学报 |遗传育种 2026 , 57 (3) : 1362 -1372
6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶3(PFKFB3)促进猪肌内前体脂肪细胞成脂分化及其互作蛋白的筛选
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李萌和陈佳仪为同等贡献作者

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李萌和陈佳仪为同等贡献作者

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陈佳仪,硕士,主要从事动物遗传育种与繁殖的研究,E-mail:

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陈佳仪,硕士,主要从事动物遗传育种与繁殖的研究,E-mail:

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李萌, 陈佳仪 , 文明昕, 韩雨琪, 钱少杰, 明祖梅, 郭晓红, 李步高
作者信息
  • 山西农业大学动物科学学院,太谷 030801
通讯作者:
李步高,主要从事动物遗传育种与繁殖的研究,E-mail:
作者简介:

李萌,博士,主要从事动物遗传育种与繁殖的研究,E-mail:

李萌和陈佳仪为同等贡献作者

陈佳仪,硕士,主要从事动物遗传育种与繁殖的研究,E-mail:

6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase 3 (PFKFB3) Promotes Adipogenic Differentiation of Porcine Intramuscular Preadipocytes and Screening of Its Interacting Proteins
Meng LI, Jiayi CHEN , Mingxin WEN, Yuqi HAN, Shaojie QIAN, Zumei MING, Xiaohong GUO, Bugao LI
Affiliations
  • College of Animal Science,Shanxi Agricultural University,Taigu 030801,China
  • Corresponding Author:
    LI Bugao, E-mail:
出版时间: 2026-03-23 doi: 10.11843/j.issn.0366-6964.2026.03.017
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旨在探究6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶3(6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3,PFKFB3)对猪肌内前体脂肪细胞增殖和成脂分化的调控作用,并筛选其互作蛋白,为解析其调控机制提供线索。本研究通过在猪肌内前体脂肪细胞中过表达/干扰PFKFB3,采用qRT-PCR检测增殖标志基因的表达变化,采用CCK-8、EdU试验检测活细胞及处于增殖期细胞数量的变化;诱导猪肌内前体脂肪细胞成脂分化后,通过qRT-PCR、油红O等技术,检测其对细胞成脂分化的调控作用;运用免疫共沉淀及液相色谱-质谱技术识别和筛选PFKFB3的互作蛋白。结果显示,过表达PFKFB3后EdU阳性细胞数量显著增加(P<0.01),细胞活力显著升高(P<0.01),且增殖相关基因CDK1、CCND1、PCNA、KI67表达显著上调(P<0.05);干扰PFKFB3则显著抑制细胞增殖(P<0.05)。成脂分化方面,过表达PFKFB3 可显著增加脂滴数量(P<0.01)和甘油三酯含量(P<0.01),并显著上调成脂关键基因PPARγ、CEBPα、CEBPβ、LPL等的表达(P<0.05);干扰PFKFB3显著抑制细胞的成脂分化(P<0.05)。此外,通过Flag标签免疫沉淀结合质谱分析共筛选到17个PFKFB3的特异性互作蛋白。GO分析表明这些蛋白主要参与ATP结合和RNA结合等过程;STRING蛋白互作网络预测显示PFKFB3与CHORDC1、ALAS1、ASCC3、KCNK2等蛋白存在相互作用。综上,PFKFB3可正向调控猪肌内前体脂肪细胞的增殖和成脂分化,并初步筛选出其潜在互作蛋白,为深入解析猪肌内脂肪沉积的分子机制及改善猪肉品质提供了新线索。

PFKFB3  /  猪  /  肌内前体脂肪细胞  /  细胞增殖  /  成脂分化  /  互作蛋白

This study aimed to investigate the regulatory role of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) in the proliferation and adipogenic differentiation of porcine intramuscular preadipocytes, screen its interacting proteins and provide clues for understanding their regulatory mechanisms. PFKFB3 was overexpressed or interfered in porcine intramuscular preadipocytes. qRT-PCR was used to detect changes in the expression of proliferation marker genes. CCK-8 and EdU assays were employed to measure changes in the number of viable cells and cells in the proliferation phase, respectively. After inducing adipogenic differentiation of porcine intramuscular preadipocytes, qRT-PCR and Oil Red O staining were used to assess its regulatory effect on adipogenic differentiation. Co-immunoprecipitation combined with liquid chromatography-tandem mass spectrometry was utilized to identify and screen proteins interacting with PFKFB3. The results showed that overexpression of PFKFB3 significantly increased the number of EdU-positive cells (P<0.01) and cell viability (P<0.01), and significantly upregulated the expression of proliferation-related genes CDK1, CCND1, PCNA, and KI67 (P<0.05). Conversely, interfering with PFKFB3 significantly inhibited cell proliferation (P<0.05). Regarding adipogenic differentiation, overexpression of PFKFB3 significantly increased the number of lipid droplets (P<0.01) and triglyceride content (P<0.01), and significantly upregulated the expression of key adipogenic genes PPARγ, CEBPα, CEBPβ, and LPL (P<0.05). Interfering with PFKFB3 significantly inhibited adipogenic differentiation (P<0.05). Furthermore, Flag-tag immunoprecipitation combined with mass spectrometry analysis identified 17 specific interacting proteins of PFKFB3. GO analysis indicated that these proteins were primarily involved in processes such as ATP binding and RNA binding. STRING protein-protein interaction network prediction revealed interactions between PFKFB3 and proteins including CHORDC1, ALAS1, ASCC3, and KCNK2. In summary, PFKFB3 positively regulates the proliferation and adipogenic differentiation of porcine intramuscular preadipocytes. Potential interacting proteins of PFKFB3 were preliminarily screened, providing new clues for in-depth analysis of the molecular mechanisms underlying porcine intramuscular fat deposition and improvement of pork quality.

PFKFB3  /  pig  /  intramuscular preadipocytes  /  cell proliferation  /  adipogenic differentiation  /  interacting proteins
李萌, 陈佳仪, 文明昕, 韩雨琪, 钱少杰, 明祖梅, 郭晓红, 李步高. 6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶3(PFKFB3)促进猪肌内前体脂肪细胞成脂分化及其互作蛋白的筛选. 畜牧兽医学报, 2026 , 57 (3) : 1362 -1372 . DOI: 10.11843/j.issn.0366-6964.2026.03.017
Meng LI, Jiayi CHEN, Mingxin WEN, Yuqi HAN, Shaojie QIAN, Zumei MING, Xiaohong GUO, Bugao LI. 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase 3 (PFKFB3) Promotes Adipogenic Differentiation of Porcine Intramuscular Preadipocytes and Screening of Its Interacting Proteins[J]. Acta Veterinaria et Zootechnica Sinica, 2026 , 57 (3) : 1362 -1372 . DOI: 10.11843/j.issn.0366-6964.2026.03.017
在全球肉类需求持续增长的背景下,肉品质日益成为决定消费者选择的核心考量。肉品质是一个复杂的性状,受许多理化特性的影响,如pH、嫩度、脂肪酸种类、肉色、肌内脂肪(intramuscular fat,IMF)含量和感官质量等[1⁃5]。IMF含量是决定猪肉抗氧化性、嫩度、风味、多汁性及总体接受度的核心因素[6⁃8]。研究发现,IMF适中的猪肉其嫩度与风味更佳。IMF不仅是风味载体,更蕴含重要的健康价值,有助于降低心血管及肌肉疾病风险[9⁃11];同时,相较于其他动物脂肪来源,IMF的胆固醇含量显著偏低[12],更契合现代消费者对营养均衡与低脂健康膳食的追求。然而,全球猪育种以提高瘦肉率和生长效率为首要目标,往往造成IMF含量减少及肉质性状下降[13]。因此,深入解析IMF沉积的调控机制,对于满足高品质猪肉消费需求、提升产业价值具有重要科学意义。
猪IMF沉积受遗传、营养、环境等多重因素影响,其中遗传因素至关重要。目前已鉴定出大量参与该过程的基因。PPARα和PPARγ参与脂肪沉积及糖异生等关键代谢通路[14⁃16]。脂肪酸结合蛋白家族成员的两个基因,FABP3和FABP4与猪肉大理石花纹及IMF含量高度相关[15,17],为脂肪沉积的关键候选基因[18⁃21]。SCD为脂肪酸合成的核心因子,其表达下调会提升脂肪组织饱和脂肪酸比例[22⁃24]。因此,阐明这些关键基因的调控网络,对于揭示IMF形成的分子机制、缓解现代育种与肉质目标的冲突、推动优质猪肉生产意义重大。然而,目前关于调控猪IMF沉积关键基因的挖掘及功能机制的研究尚不完善,亟待深入探索。
鉴于IMF沉积对猪肉品质的核心作用,解析其关键调控因子及分子机制已成为研究焦点。6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶3(6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3,PFKFB3)是糖酵解通路中的双功能代谢枢纽,其激酶结构域催化果糖-6-磷酸(f6P)合成果糖-2,6-二磷酸(f2,6BP),也可经磷酸酶结构域水解f2,6BP[25]。F2,6BP是磷酸果糖激酶-1的强效变构激活剂,同时会抑制糖异生途径的关键酶—果糖-1,6-二磷酸酶(fructose-1,6-bisphosphatase,FBPase1)[26]。此外,PFKFB3在脂肪代谢中扮演关键角色。研究表明,小鼠脂肪组织特异性过表达PFKFB3可显著增加脂肪沉积,在一定程度上使其胰岛素敏感性得到改善[27]。其机制涉及促进脂肪细胞糖酵解及糖酵解驱动的脂肪生成,并可能介导PPARγ激活的抗炎与抗糖尿病效应[28]。相反,干扰PFKFB3后,则导致脂质积累减少、炎症因子(TNFα、IL-6)水平升高及胰岛素信号受损[29]。这些证据共同表明,PFKFB3是调控脂肪细胞代谢稳态与脂质储存的核心分子。然而,PFKFB3在猪肌内脂肪沉积中的具体调控作用及分子机制仍鲜有报道。
本研究以猪原代肌内脂肪细胞为模型,通过功能获得与缺失试验,系统探究PFKFB3对细胞成脂的作用;机制层面,借助免疫共沉淀-质谱联用技术筛选其互作蛋白,初步揭示PFKFB3的调控机制。本研究结果可为解析猪肌内脂肪沉积的分子机制提供新视角。
选取3头初生杜×长×大仔猪,用于猪肌内前体脂肪细胞的分离。
胰酶和青链霉素购自美国Sigma公司;胎牛血清购自美国Gibco公司;胰岛素、红细胞裂解液和饱和油红O染液购自中国Solarbio公司;siRNA和过表达载体、Lipofectamine 3000 Reagent购自上海汉恒基因科技有限公司;CCK-8试剂盒和EdU试剂盒购自亚科因生物技术有限公司;快速银染试剂盒、Flag标签蛋白免疫沉淀试剂盒、BCA蛋白浓度测定试剂盒购自上海碧云天生物技术有限公司。
仔猪放血处死后依次使用灭菌蒸馏水及75%乙醇进行体表消毒,采集背最长肌组织样本,将样本置于75%乙醇溶液中浸泡消毒,随后转移至含4%双抗的PBS中进行漂洗。随后去除组织中的血管以及结缔组织,将处理干净的组织剪碎后加入消化液,恒温振荡培养箱(37 ℃,120 r·min-1)中进行消化处理,持续60 min至组织块消化到看不出形状,终止消化后进行梯度过滤(70 μm和40 μm细胞筛),经过离心后去除上清保留沉淀(1 500 r·min-1,10 min)。用红细胞裂解液重悬细胞,静置2 min,加入10 mL无血清DMEM/F12培养基,相同转速离心5 min后,弃上清液,重复清洗一次。最终使用含有10%胎牛血清的DMEM/F12完全培养基重悬沉淀,置于预处理培养器皿中,于37 ℃、5% CO2培养箱中进行原代培养,4 h后换液。待培养瓶中的细胞长满后消化重新接种于6孔板当中,长满后换分化培养基,4 d后换维持培养基。
待细胞密度为60%时进行转染。转染前更换为无血清DMEM/F12基础培养基,参照转染试剂Lipofectamine 3000的说明书进行转染,试验设计每个处理包含3个重复,转染48 h后检测过表达和干扰效率。
在细胞培养板中,对转染过表达和干扰后的细胞,加入与培养液等体积的EdU工作液孵育4 h,使用4%多聚甲醛对细胞进行固定,固定时间为20 min,随后采用0.1%非离子型表面活性剂Triton X-100孵育30 min,随后置于避光环境中加入预先配置好的Click反应液,孵育40 min,最后加入Hoechst,孵育20 min,PBS清洗3次后,在荧光倒置显微镜下观察和拍照。加入CCK-8试剂后,在不同时间检测450 nm波长下的吸光度。
成脂诱导分化8 d后,进行油红O染色及胞内甘油三酯(Triglycerides,TG)含量测定。油红O染色:除去培养基,PBS洗涤细胞两次,固定液固定30 min;60%异丙醇处理;油红O染液室温避光染色15 min;移除染液,60%异丙醇漂洗至背景清晰,蒸馏水充分洗涤;Mayer苏木素复染细胞核,弱碱性缓冲液返蓝;蒸馏水覆盖,光学显微镜下观察拍照。同时,定量脂质积累,裂解细胞,取上清。采用商品化TG测定试剂盒,酶标仪检测吸光度计算TG含量。
在细胞中加入裂解液,使用细胞刮板收集蛋白样品。根据蛋白样品与磁珠的配比(1 000 μL样品对应40 μL磁珠悬液),将其混合并转移至低温水平振荡仪上,4 ℃持续孵育16 h。随后将其放置于磁力架上10 s,移除1.5 mL离心管中的液体并添加裂解缓冲液,进行磁珠洗涤。根据磁珠初始体积按1∶5(40 μL磁珠对应200 μL洗脱液)加入3×Flag多肽洗脱缓冲液,充分混匀后置于4 ℃孵育2 h,随后放置于磁力架上10 s,收集含有目标蛋白复合物的上清液,立即分装至预冷的离心管中,于-80 ℃保存。
制备分离胶和浓缩胶,将洗脱的样品与5×SDS LoadingBuffer混合于100 ℃煮沸,然后将样品缓慢加入胶孔中,80 V恒压电泳直至样品进入分离胶;随后,120 V恒压电泳。银染按照快速银染试剂盒说明书操作。
银染完成后,对差异表达的条带进行切割,质谱分析。使用LC-MS/MS分析方法,用MaxQuant1.6.14检索相应的数据库,鉴定其中的蛋白,并进一步对蛋白所属的所有GO条目进行汇总分析,针对所筛选出的互作蛋白采用STRING构建蛋白相互作用网络关系图。
按说明书使用Trizol试剂提取背最长肌组织中和细胞的总RNA,样本检测合格之后-80 ℃保存。用PrimeScrip RT试剂盒合成cDNA,使用SYBR®PrimeScriptTM RT-PCR试剂盒进行定量qRT-PCR,反应的总体积为10 μL:引物的上、下游各需0.3 μL,cDNA需4.4 μL,使用q-Mix补充至10 μL。反应程序为94 ℃ 30 s;94 ℃ 5 s,60 ℃ 15 s,72 ℃ 10 s,45个循环。引物序列见表1,以18S rRNA为参照基因,数据结果使用2-∆∆Ct计算。
数据结果使用SPSS 27.0软件独立样本T检验进行显著性分析,两组间比较满足正态分布时采用独立样本t检验,多组间比较采用单因素方差分析(ANOVA)。采用Graphpad Prism 9.5软件作图。P<0.05被认为具有统计学显著性。
当细胞接近长满时,诱导成脂分化,2 d后显微镜下观察开始出现小脂滴,集中于细胞核周围(图1A);第8天小脂滴逐渐汇合成大的脂滴,其着色呈橘红色,形态大小不一,证实可分化为成熟脂肪细胞(图1B)。
通过在猪肌内前体脂肪细胞中过表达PFKFB3,探究其在细胞增殖中的作用。结果表明,与OE-NC组相比,OE-PFKFB3组PFKFB3的表达水平显著升高(P<0.01,图2A)。EdU染色结果显示,在猪肌内前体脂肪细胞中过表达PFKFB3后阳性细胞数量显著高于OE-NC组(P<0.01,图2B-C)。CCK-8结果显示过表达PFKFB3后细胞活力显著升高(P<0.01,图2D)。qRT-PCR结果显示,相较于OE-NC组,OE-PFKFB3组中CDK1、CCND1、PCNA和KI67的表达水平显著提高(P<0.05,图2E)。以上试验结果共同表明,过表达PFKFB3可促进细胞增殖。
干扰PFKFB3发现添加si-PFKFB3-1极显著下调PFKFB3的表达(P<0.01,图3A),在随后的试验中使用si-PFKFB3-1并标记为si-PFKFB3。EdU染色结果显示,转染si-PFKFB3后阳性细胞数量显著低于si-NC组(P<0.01,图3B-C)。CCK-8结果显示干扰PFKFB3后细胞活力显著降低(P<0.05,图3D)。qRT-PCR结果显示,相较于si-NC组,si-PFKFB3组中增殖标志基因CDK1,CCND1、PCNA和KI67的mRNA表达水平显著显著降低(P<0.01,图3E)。综上所述,干扰PFKFB3抑制猪肌内前体脂肪细胞增殖。
与OE-NC组相比,油红O染色发现OE-PFKFB3组脂滴数量明显增多(P<0.01,图4A-B),过表达PFKFB3后细胞中的TG含量极显著上升(P<0.01,图4C)。qRT-PCR结果表明,与OE-NC组相比,过表达PFKFB3后PPARγ、CEBPα、CEBPβ、LPL等成脂标志基因表达量显著升高(P<0.05,图4D)。
在猪肌内前体脂肪细胞中转染si-PFKFB3并诱导细胞成脂分化8 d检测细胞成脂情况。油红O染色发现干扰PFKFB3后脂滴明显减少(P<0.01,图5A-B);肌内前脂肪细胞中的TG含量极显著下降(P<0.01,图5C)。qRT-PCR结果表明,与si-NC组相比,干扰PFKFB3后PPARγ、CEBPα等成脂标志基因表达量显著降低(P<0.05,图5D)。
细胞蛋白提取后通过Flag标签蛋白免疫沉淀获得的蛋白设置为空白对照、IgG和PFKFB3组,通过SDS-PAGE电泳后银染发现PFKFB3 pull-down的蛋白中存在特异性表达的蛋白(图6A)。质谱结果表明,PFKFB3-NC组和PFKFB3-OE组共富集到207个蛋白,其中,PFKFB3-NC组鉴定到36个特异互作蛋白,PFKFB3-OE组鉴定到17个特异互作蛋白(图6B)其主要参与ATP结合和RNA结合等生物过程(图6C);采用STRING构建PFKFB3蛋白相互作用网络,发现PFKFB3与CHORDC1、ALAS1等存在较强的互作关系(图6D)。
IMF是调控猪肉品质的核心要素,其含量水平与化学组成是影响猪肉嫩度、风味形成及营养价值的关键变量[8,30]。通常,较高IMF含量赋予猪肉更佳的嫩度和多汁性。这主要归因于IMF填充于肌纤维间隙,降低肌纤维束紧密度,从而改善肉质的口感和咀嚼特性。研究表明,高IMF含量的猪肉在烹饪过程中保水能力更强,有助于维持肉质鲜嫩[31]。此外,IMF对风味形成至关重要。热加工时,其不饱和脂肪酸组分发生氧化反应,降解产生醛类、酮类和醇类等一些挥发性风味化合物,对肉品特征性香气的形成与整体风味的增强至关重要[32,33]。值得注意的是,猪肌内脂肪中饱和脂肪酸比例相对较低,这不仅有助于保持良好的风味,还可能降低心血管及肌肉疾病风险[9⁃11]。本课题组前期研究发现,纳米维生素A能显著提高仔猪生长速度并促进肌内脂肪沉积。基于此,通过多组学联合分析确定PFKFB3为调控猪肌内脂肪沉积的一个关键候选基因。
作为糖酵解途径的核心限速酶,PFKFB3通过催化f2,6BP的转化,不仅精细调节糖酵解过程,同时深刻影响细胞周期动态[34]。PFKFB3可调控细胞周期关键因子,如促进Cyclin与其依赖激酶的活性,并抑制p27表达,增加S期细胞的数量[35]。本研究证明,PFKFB3显著促进细胞增殖,与上述结果一致。脂肪生成是一个受多阶段、多信号通路和转录因子精密调控的复杂生物学过程[36⁃38],PFKFB3在此过程中发挥着重要的作用。既往研究指出,PFKFB3可增强脂肪细胞的糖酵解和脂质生成能力[39]。本研究证明,PFKFB3可促进猪肌内前体脂肪细胞的成脂分化。
本研究通过互作蛋白筛选,发现PFKFB3可能与CHORDC1、ALAS1、ASCC3及KCNK2等蛋白存在相互作用。其中,富含半胱氨酸和组氨酸的结构域蛋白1(CHORDC1)的功能尤为值得关注。作为HSP90的重要共伴侣分子,CHORDC1可参与细胞应激响应、能量平衡、蛋白质折叠与组装等关键过程[40]。其广泛互作能力使其在细胞周期调控和免疫应答等生物学过程中发挥重要作用[41]。PFKFB3是糖酵解通路中的核心调控因子,是细胞能量代谢的关键枢纽[42,43]。CHORDC1对HSP90活性的调节高度依赖于核苷酸状态,尤其在ADP/ATP比例升高时作用更为显著[40]。基于此,我们提出一种潜在的作用模式:PFKFB3可能通过其强大的促糖酵解能力,维持细胞内充足ATP供应,优化ADP/ATP比例,从而间接影响CHORDC1-HSP90复合物的构象与功能活性,最终调控其介导的蛋白质折叠、应激响应及能量感知等过程。这种PFKFB3-CHORDC1的功能性耦联,可能为理解糖酵解代谢流如何影响蛋白质稳态及细胞应激适应提供新视角,尤其在猪肌内前体脂肪细胞的成脂分化微环境中。此外,氨基乙酰丙酸合酶1(ALAS1)是血红素生物合成途径的限速酶,主要参与血红素的生物合成,在血红素合成的负反馈调节中发挥作用[44],推测,PFKFB3可能通过调节能量代谢状态影响血红素合成效率,进而调控线粒体功能及细胞色素相关酶的活性,最终参与脂质代谢的精细调控。激活信号协整合复合体亚基3(ASCC3)是激活信号协整合复合体的核心解旋酶组分,其在修复烷基化DNA损伤、维护基因组稳定性中发挥关键作用[45],我们推测,在脂肪细胞快速增殖与分化过程中,FKFB3-ASCC3互作可能旨在高效修复复合体,确保脂代谢相关的关键基因在转录程序顺利进行,从而促进成脂分化。钾通道蛋白KCNK2(TREK-1)是一种双孔钾通道,在多种细胞类型中被证实具有抑制细胞增殖的功能[46],我们推测,PFKFB3可能通过调节KCNK2的通道活性,进而共同调控前体脂肪细胞由增殖转向分化,为脂质沉积调控提供新视角,并揭示离子通道在代谢中的非经典功能。PFKFB3是过氧化物酶体增殖物激活受体γ(PPARγ)的靶基因[29],推测PFKFB3可能是PPARγ行使功能的关键下游,通过驱动糖酵解代谢流,为PPARγ介导的脂肪生成提供必需的能量和底物。这些发现深度揭示了糖酵解代谢枢纽PFKFB3对猪肌内脂肪沉积的调控作用,并初步研究了其调控机制,为解析脂肪细胞发育的代谢调控提供了新视角。
本研究在猪肌内前体脂肪细胞模型中系统阐明了PFKFB3通过促进细胞增殖和驱动成脂分化双重机制调控肌内脂肪沉积,同时通过互作蛋白筛选鉴定出CHORDC1、ALAS1等新型互作因子,为解析PFKFB3调控脂肪细胞代谢的分子机制提供了新线索。
  • 山西省重点研发计划项目(202302140601005)
  • 山西省生猪育种联合攻关项目(NYGG21)
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2026年第57卷第3期
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doi: 10.11843/j.issn.0366-6964.2026.03.017
  • 接收时间:2025-06-27
  • 首发时间:2026-09-16
  • 出版时间:2026-03-23
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  • 收稿日期:2025-06-27
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山西省重点研发计划项目(202302140601005)
山西省生猪育种联合攻关项目(NYGG21)
作者信息
    山西农业大学动物科学学院,太谷 030801

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李步高,主要从事动物遗传育种与繁殖的研究,E-mail:
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2种不同金属材料的力学参数
科
Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)
属
Genus
种数
Number of
species
占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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