过表达HMOX1基因对无量山乌骨鸡原代前脂肪细胞增殖的影响

doi:10.16431/j.cnki.1671-7236.2026.01.032

摘要/Abstract

摘要：

目的构建无量山乌骨鸡血红素加氧酶1（heme oxygenase 1，HMOX1）基因过表达载体，并转染鸡原代前脂肪细胞，探究过表达HMOX1基因对鸡原代前脂肪细胞增殖的影响。方法取1日龄无量山乌骨鸡雏鸡皮下脂肪组织，提取总RNA并反转录成cDNA，扩增HMOX1基因CDS区，使用质粒pcDNA3.1-mRFP构建过表达载体pcDNA3.1-HMOX1-mRFP，并进行酶切和测序鉴定；通过转染试剂将pcDNA3.1-HMOX1过表达载体转染鸡原代前脂肪细胞中并检测过表达效率；利用CCK-8法检测细胞活力，流式细胞术检测细胞周期；利用实时荧光定量PCR和Western blotting检测增殖相关基因P21、CDK1、PCNA mRNA和蛋白表达量。结果酶切和测序结果表明，pcDNA3.1-HMOX1-mRFP过表达载体构建成功。实时荧光定量PCR和Western blotting检测结果显示，与对照组相比，转染pcDNA3.1-HMOX1-mRFP后细胞中HMOX1基因mRNA和蛋白表达量均极显著上调（P<0.01），表明鸡原代前脂肪细胞中成功过表达HMOX1。CCK-8检测结果显示，与对照组相比，转染12 h时过表达HMOX1组细胞活力显著上升（P<0.05），转染36和48 h时细胞活力极显著下降（P<0.01）。流式细胞术检测结果显示，与对照组相比，12 h时过表达HMOX1组S期的细胞数量极显著增加（P<0.01），G1期的细胞数量极显著降低（P<0.01）；48 h时S期的细胞数量显著降低（P<0.05）。增殖相关基因检测结果显示，两组间细胞中P21、CDK1基因mRNA和蛋白表达量无显著差异（P>0.05）；过表达HMOX1组细胞中PCNA基因mRNA和蛋白表达量均显著低于对照组（P<0.05）。结论本研究成功构建HMOX1基因过表达载体，转染无量山乌骨鸡原代前脂肪细胞后抑制了细胞增殖。试验结果可为进一步研究HMOX1对无量山乌骨鸡原代前脂肪细胞的分化作用提供理论指导。

关键词: 无量山乌骨鸡; HMOX1基因; 前脂肪细胞; 增殖

Abstract:

Objective This experiment aimed to construct an overexpression vector of heme oxygenase 1 （HMOX1） gene in Wuliangshan Black-boned chickens and transfect it into chicken primary preadipocytes， and investigate the effect of HMOX1 gene overexpression on the proliferation of chicken primary preadipocyte. Method Collect the subcutaneous fat tissue from 1-day-old chicks of Wuliangshan Black-boned chicken， extract total RNA and reverse transcribe it into cDNA， and amplify the CDS region of HMOX1 gene. The overexpression vector pcDNA3.1-HMOX1-mRFP was constructed using the plasmid pcDNA3.1-mRFP， followed by restriction enzyme digestion and sequencing for identification. The pcDNA3.1-HMOX1 overexpression vector was transfected into chicken primary preadipocytes by using transfection reagents， and the overexpression efficiency was detected. Cell viability was measured using CCK-8 method， and the cell cycle was analyzed by flow cytometry. Real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expression levels of proliferation-related genes （P21， CDK1 and PCNA）. Result Restriction enzyme digestion and sequencing indicated that the pcDNA3.1-HMOX1-mRFP overexpression vector was successfully constructed. Real-time quantitative PCR and Western blotting test results showed that， compared with control group， the mRNA and protein expression of HMOX1 gene in cells transfected with pcDNA3.1-HMOX1-mRFP were extremely significantly upregulated （P<0.01）， demonstrated that HMOX1 was successfully overexpressed in chicken primary preadipocytes. CCK-8 assay results showed that compared with control group， the cell viability of HMOX1 overexpression group significantly increased at 12 hours after transfection （P<0.05）， and decreased extremely significantly at 36 and 48 hours after transfection （P<0.01）. The results of flow cytometry test showed that，compared with control group， the number of cells in the S phase in HMOX1 overexpression group increased extremely significantly at 12 hours （P<0.01）， while the number of cells in the G1 phase decreased extremely significantly （P<0.01）. The number of cells in the S phase at 48 hours was significantly reduced （P<0.05）. Detection results of proliferation-related genes showed that there was no significant difference in the mRNA and protein expression of P21 and CDK1 genes in the cells between the two groups （P>0.05）. The mRNA and protein expression of PCNA gene in overexpression group were significantly lower than those in control group （P<0.05）. Conclusion The HMOX1 gene overexpression vector was successfully constructed. After transfection of the original preadipocytes of Wuliangshan Black-boned chicken， cell proliferation was inhibited. The experimental results could provide theoretical guidance for further study of the effect of HMXO1 on the differentiation of primary preadipocytes of Wuliangshan Black-boned chicken.

Key words: Wuliangshan Black-boned chicken; HMOX1 gene; preadipocyte; proliferation

中图分类号:

S831.2

杨平远, 殴小嫚, 殴正淼, 陈粉粉. 过表达HMOX1基因对无量山乌骨鸡原代前脂肪细胞增殖的影响[J]. 中国畜牧兽医, 2026, 53(1): 359-367.

YANG Pingyuan, OU Xiaoman, OU Zhengmiao, CHEN Fenfen. Effect of HMOX1 Gene Overexpression on the Proliferation of Primary Preadipocyte in Wuliangshan Black-boned Chicken[J]. China Animal Husbandry & Veterinary Medicine, 2026, 53(1): 359-367.

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参考文献 27

[1]	SANADA Y， TAN S J O， ADACHI N， et al. Pharmacological targeting of heme oxygenase-1 in osteoarthritis ［J］. Antioxidants， 2021， 10（3）： 419.
[2]	DRUMMOND G S， BAUM J， GREENBERG M， et al. HO-1 overexpression and underexpression： Clinical implications［J］. Archives of Biochemistry and Biophysics， 2019， 673： 108073.
[3]	YAO H， PETERSON A L， LI J， et al. Heme oxygenase 1 and 2 differentially regulate glucose metabolism and adipose tissue mitochondrial respiration： Implications for metabolic dysregulation［J］. International Journal of Molecular Sciences， 2020， 21（19）： 7123.
[4]	RYTER S W. Heme oxgenase-1， a cardinal modulator of regulated cell death and inflammation［J］. Cells， 2021， 10（3）： 515.
[5]	SORRENTI V. Editorial of special issue “protective and detrimental role of heme oxygenase-1”：2021［J］. International Journal of Molecular Sciences， 2023， 24（5）： 4386.
[6]	DUNN L L， MIDWINTER R G， NI J， et al. New insights into intracellular locations and functions of heme oxygenase-1［J］. Antioxid Redox Signal， 2014， 20（11）： 1723-1742.
[7]	SHI J， SU M. HMOX1 participates in pre-eclampsia by regulating the proliferation， apoptosis， and angiogenesis modulation potential of mesenchymal stem cells via VEGF［J］. Biochemical Genetics， 2024， 62（2）： 1248-1262.
[8]	宋佳伦，王心正，吴瑾，等.HMOX1在低氧条件下对胶质瘤干细胞的作用［J］.科学技术与工程，2017，17（31）： 193-198.
	SONG J L， WANG X Z， WU J， et al. Effect of HMOX1 on glioma stem cells under hypoxia［J］. Science Technology and Engineering， 2017， 17（31）： 193-198. （in Chinese）
[9]	CHEN C， SHEN H， HUANG Q， et al. The circular RNA CDR1as regulates the proliferation and apoptosis of human cardiomyocytes through the miR-135a/HMOX1 and miR-135b/HMOX1 axes［J］. Genetic Testing and Molecular Biomarkers， 2020， 24（9）： 537-548.
[10]	SINGH S P， GRANT I， MEISSNER A， et al. Ablation of adipose-HO-1 expression increases white fat over beige fat through inhibition of mitochondrial fusion and of PGC1α in female mice［J］. Hormone Molecular Biology and Clinical Investigation， 2017， 31（1）： 28763300.
[11]	WAGNER G， LINDROOS-CHRISTENSEN J， EINWALLNER E， et al. HO-1 inhibits preadipocyte proliferation and differentiation at the onset of obesity via ROS dependent activation of Akt2［J］. Scientific Reports， 2017， 7： 40881.
[12]	LI Q， SPALDING K L. The regulation of adipocyte growth in white adipose tissue［J］. Frontiers in Cell and Developmental Biology， 2022， 10： 1003219.
[13]	CAO Y， XING Y， GUAN H， et al. Genomic insights into molecular regulation mechanisms of intramuscular fat deposition in chicken［J］. Genes， 2023， 14（12）： 2197.
[14]	殴小嫚，刘明欢，吴芸，等.无量山乌骨鸡HMOX1基因克隆、生物信息学分析及组织表达研究［J］.中国畜牧兽医， 2023， 50（11）： 4381-4391.
	OU X M， LIU M H， WU Y， et al. Cloning， bioinformatics and tissue expression analysis of HMOX1 gene in Wuliangshan Sooty chickens［J］. China Animal Husbandry & Veterinary Medicine， 2023， 50（11）： 4381-4391.（in Chinese）
[15]	SHI H， DUAN X， DONG J， et al. RNA-Seq combined network pharmacology reveals that Fu-Gan-Wan （FGW） inhibits liver fibrosis via NF-κB/CCL2/CCR2 and lipid peroxidation via Nrf2/HMOX1 signaling pathway［J］. Journal of Ethnopharmacology， 2024， 326： 117963.
[16]	YUAN X， LI L， ZHANG Y， et al. Heme oxygenase 1 alleviates nonalcoholic steatohepatitis by suppressing hepatic ferroptosis［J］. Lipids in Health and Disease， 2023， 22（1）： 99.
[17]	TSAI Y C， WANG C W， WEN B Y， et al. Involvement of the p62/Nrf2/HO-1 pathway in the browning effect of irisin in 3T3-L1 adipocytes［J］. Molecular and Cellular Endocrinology， 2020， 514： 110915.
[18]	PETERSON S J， RUBINSTEIN R， FAROQUI M， et al. Positive effects of heme oxygenase upregulation on adiposity and vascular dysfunction： Gene targeting vs. pharmacologic therapy［J］. International Journal of Molecular Sciences， 2019， 20（10）： 2514.
[19]	黄昭伟，朱昭炜，许澍洽，等.诱导时间对脂肪干细胞来源的类许旺细胞增殖率的影响［J］.中华显微外科杂志， 2019， 42（2）： 150-154.
	HUANG Z W， ZHU Z W， XU S Q， et al. Investigation effect of induction time on proliferation rate of induced Schwann-like cells from adipose derived stem cells［J］. Chinese Journal of Microsurgery，2019， 42（2）： 150-154. （in Chinese）
[20]	WU Y D， LI M， LIAO X， et al. Effects of storage culture media， temperature and duration on human adipose-derived stem cell viability for clinical use［J］. Molecular Medicine Reports， 2019， 19（3）： 2189-2201.
[21]	席凤雪.miR-214-3p对3T3-L1前体脂肪细胞分化的作用及机制研究［D］.杨凌：西北农林科技大学，2019.
	XI F X. The effect of miR-214-3p on the differentiation of 3T3-L1 preadipocytes and the mechanism of action［D］. Yangling： Northwest A&F University， 2019. （in Chinese）
[22]	XIAO H， TIAN M， GE J， et al. The role of CDK1 siRNA interference in cell cycle and cell apoptosis［J］. Frontiers of Medicine in China， 2009， 3（4）： 384-389.
[23]	NAAZ A， HOLSBERGER D R， IWAMOTO G A， et al. Loss of cyclin-dependent kinase inhibitors produces adipocyte hyperplasia and obesity［J］. The FASEB Journal， 2004， 18（15）： 1925-1927.
[24]	QIMUGE N， HE Z， QIN J， et al. Overexpression of DNMT3A promotes proliferation and inhibits differentiation of porcine intramuscular preadipocytes by methylating p21 and PPARg promoters［J］. Gene， 2019， 696： 54-62.
[25]	LO Y H， HO P C， CHEN M S， et al. Phosphorylation at tyrosine 114 of proliferating cell nuclear antigen （PCNA） is required for adipogenesis in response to high fat diet［J］. Biochemical and Biophysical Research Communications， 2013， 430（1）： 43-48.
[26]	MORENO-NAVARRETE J M， ORTEGA F， RODRÍGUEZ A， et al. HMOX1 as a marker of iron excess-induced adipose tissue dysfunction， affecting glucose uptake and respiratory capacity in human adipocytes［J］. Diabetologia， 2017， 60（5）： 915-926.
[27]	ABRAHAM N G， SINGH S P， BELLNER L， et al. HMOX1 activation reprograms white fat to beige adipose tissue through recruitment of Cyp2C44-derived EET， pAMPK-PGC1α that enhances mitochondrial Mfn2 and Opa1［J］. Hypertension， 2016， 68（）： A079.

基因 Genes	引物序列 Primer sequences （5′→3′）	产物长度 Product length/bp	GenBank登录号 GenBank accession No.
HMOX1	F：GAGCCAGGAGAACGGTCAT R：CCTGCTTGTCCTCTCACTCT	115	NC_052532.
β-actin	F：GAGAAATTGTGCGTGACATCA R：CCTGAACCTCTCATTGCCA	152	NC_006101.5
CDK1	F：TGCCATTCAAGACGAGTTCTG R：AGGAGTGGAATAA	198	NM_205314.1
P21	F：GAAGAGTTGTCCACGATAAGC R：TTCCAGTCCTCCTCA	247	AF513031.1
PCNA	F：ATGCGGATACGTTGGCTCTA R：CAATGTGGCTGAG	180	NM_204170.2