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中國精品科技期刊2020

異麥芽酮糖減少小鼠肝臟脂肪堆積的關鍵基因篩選與驗證

何秋玲 張彩平 桂靜 劉泳杏 季久秀 周波 張震

何秋玲,張彩平,桂靜,等. 異麥芽酮糖減少小鼠肝臟脂肪堆積的關鍵基因篩選與驗證[J]. 食品工業科技,2022,43(16):1?8. doi:  10.13386/j.issn1002-0306.2021110090
引用本文: 何秋玲,張彩平,桂靜,等. 異麥芽酮糖減少小鼠肝臟脂肪堆積的關鍵基因篩選與驗證[J]. 食品工業科技,2022,43(16):1?8. doi:  10.13386/j.issn1002-0306.2021110090
HE Qiuling, ZHANG Caiping, GUI Jing, et al. Screening and Verification of Key Genes for Isomaltulose Reducing Liver Fat Accumulation in Mice[J]. Science and Technology of Food Industry, 2022, 43(16): 1?8. (in Chinese with English abstract). doi:  10.13386/j.issn1002-0306.2021110090
Citation: HE Qiuling, ZHANG Caiping, GUI Jing, et al. Screening and Verification of Key Genes for Isomaltulose Reducing Liver Fat Accumulation in Mice[J]. Science and Technology of Food Industry, 2022, 43(16): 1?8. (in Chinese with English abstract). doi:  10.13386/j.issn1002-0306.2021110090

異麥芽酮糖減少小鼠肝臟脂肪堆積的關鍵基因篩選與驗證

doi: 10.13386/j.issn1002-0306.2021110090
基金項目: 廣西自然科學基金(2020GXNSFBA297026);廣西高校中青年教師基礎能力提升項目(2020KY12007);山東自然科學基金(ZR2019PC034);國家級大學生創新創業訓練項目(202010601028)。
詳細信息
    作者簡介:

    何秋玲(1997?),女,碩士研究生,研究方向:藥物研發與轉化,E-mail:1467925313@qq.com

    通訊作者:

    周波(1988?),男,博士,講師,研究方向:藥物化學,E-mail:zb090900021@163.com

    張震(1992?),男,博士,講師,研究方向:生物學,E-mail:zhenzhang@glmc.edu.cn

  • 中圖分類號: TS241

Screening and Verification of Key Genes for Isomaltulose Reducing Liver Fat Accumulation in Mice

  • 摘要: 為篩選和驗證異麥芽酮糖減少小鼠肝臟脂肪堆積的關鍵基因,本研究利用Limma軟件包對異麥芽酮糖和蔗糖飼喂22周的雄性小鼠肝臟轉錄組數據進行差異表達分析(GSE54723,n=14),共篩選出49個DEGs,通過Metascape軟件對DEGs進行GO功能富集分析和KEGG信號通路分析,使用Cytoscape軟件和WGCNA軟件包構建基因共表達網絡,取重疊基因搜尋關鍵節點基因,挖掘得到10個關鍵節點基因Pnliprp1、Prss2、Clps、Tff2、Pnlip、Ctrl、Cpa1、Cel、Dmbt1、Vil1,基因功能分析表明Pnlip、Pnliprp1、Cel、Clps與脂質代謝相關。qPCR結果顯示,異麥芽酮糖組肝臟組織中這4個與脂質代謝相關基因的表達量顯著高于蔗糖組,通過比較正常人和非酒精性脂肪肝患者肝臟組織轉錄組數據中上述基因的表達量(GSE163211,n=318),發現正常人Pnlip、Pnliprp1、Cel、Clps基因表達量亦顯著(P<0.05) 高于病例組。本研究揭示了異麥芽酮糖通過上調Pnlip、Pnliprp1、Cel、Clps的合成促進脂肪分解從而降低肝臟脂質的積累,為異麥芽酮糖影響肝臟脂質代謝的分子機制研究提供了理論支撐。
  • 圖  1  異麥芽酮糖與蔗糖處理組的差異基因表達火山圖(A)、通路富集圖(B)和柱狀圖(C)

    Figure  1.  Volcano plot (A), pathway enrichment plot (B) and histogram (C) of differentially expressed genes between isomaltulose and sucrose treatment groups

    注:A:紅色圓點代表上調基因,綠色三角形代表下調基因;B:綠色圓點代表基因富集的生物學過程;C:粉紅色柱狀圖代表上調基因和差異倍數,綠色斜線柱狀圖代表下調基因和差異倍數。

    圖  2  異麥芽酮糖與蔗糖組的差異基因互作網絡圖

    Figure  2.  Differential gene interaction network of isomaltulose and sucrose groups

    注:A:使用Cytoscape軟件構建的蛋白質互作網絡圖;B:使用WGCNA軟件構建的基因互作網絡圖。

    圖  3  異麥芽酮糖減少小鼠肝臟脂肪堆積的關鍵基因的驗證

    Figure  3.  Validation of key genes of isomaltulose reducing liver fat accumulation in mice

    注:10個關鍵節點基因的互作圖(A);Pnliprp1,Pnlip,Cel,Clps基因在小鼠肝臟中的qPCR驗證圖(B);*為P<0.05,**為P<0.01;空白對照組(C)、異麥芽酮糖(D)和蔗糖(E)飼喂組小鼠肝臟組織HE染色圖(40×)。

    圖  4  PNLIP、PNLIPRP1、CEL、CLPS基因在正常人和NAFLD患者肝臟組織中的基因表達量圖

    Figure  4.  PNLIP,PNLIPRP1,CEL,CLPS gene expression in liver tissues of normal people and NAFLD patients

    注:*為P<0.05;**為P<0.01。

    表  1  本研究中所用的引物列表

    Table  1.   List of primers used in this study

    序號引物名稱引物序列擴增基因名稱擴增片段(bp)退火溫度(℃)
    1Pnlip-FCTGGGAGCAGTAGCTGGAAGPnlip13361
    Pnlip-RAGCGGGTGTTGATCTGTGC
    2Pnliprp1-FGGAAGACTCGGTTCATCATCCAPnliprp17461
    Pnliprp1-RCACCCACTACTCGCACATTGT
    3Clps-FGAACAGTATGCAGTGTAAGAGCAClps20861
    Clps-RGCAGATGCCATAGTTGGTGTTG
    4Cel-FCGCCTGGAGGTTCTATTTCTTGCel9561
    Cel-RTCCACGAAACCGCCTTCTG
    5Actb-FGGCTGTATTCCCCTCCATCGβ-actin15461
    Actb-RCCAGTTGGTAACAATGCCATGT
    下載: 導出CSV

    表  2  異麥芽酮糖和蔗糖飼喂組間小鼠肝臟組織差異表達的基因及其功能

    Table  2.   Differentially expressed genes and their functions in liver tissue of mice fed with isomaltulose and sucrose

    基因英文簡稱基因英文全稱基因中文名基因功能P
    Reg1Regenerating Family Member 1α再生家族成員1α與胰島細胞的再生和糖尿病的發生有關1.24×10?6
    Zg16Zymogen Granule Protein 16酶原顆粒蛋白16運輸蛋白質2.05×10?6
    Prap1Proline Rich Acidic Protein 1富含脯氨酸的酸性
    蛋白質1
    脂質結合蛋白,通過促進MTTP介導的脂質轉移
    (主要是甘油三酯和磷脂)和MTTP介導的apoB
    脂蛋白的組裝和分泌來促進脂質吸收
    2.63×10?6
    Pnliprp1Pancreatic Lipase Related Protein 1胰脂肪酶相關蛋白1參與甘油三酯的消化,脂蛋白代謝和新陳代謝8.62×10?6
    Ela2Elastase彈性蛋白酶激活絲氨酸型內肽酶活性,作用于多個過程的上游或內部,包括對抗原刺激的急性炎癥反應以及對脂多糖的反應1.25×10?5
    PnlipPancreatic Lipase胰三酰甘油脂肪酶在脂肪代謝中起重要作用4.48×10?5
    ClpsColipase脂肪酶胰腺脂肪酶的一個輔助因子,使脂肪酶能夠將自己錨定在脂質-水界面上,發揮消化脂質功能6.40×10?5
    Ly64Lymphocyte Antigen 64淋巴細胞抗原64催化蛋白質同源二聚化活性,參與胃腸上皮的維持1.74×10?4
    Tff2Trefoil Factor 2三葉因子2抑制胃腸道蠕動和胃酸分泌2.30×10?4
    Rnase1Ribonuclease A Family Member 1核糖核酸酶A
    家族成員1
    催化裂解嘧啶核苷酸3'側的RNA的內切酶2.90×10?4
    SisSimian Sarcoma猿猴肉瘤與血小板衍生生長因子B亞單位同源,在胚胎發育、細胞增殖、細胞遷移、存活和趨化性的調節中起重要作用4.01×10?4
    Vil1Villin 1絨毛蛋白1肌動蛋白修飾蛋白,調節微絨毛肌動蛋白絲的重組4.20×10?4
    Socs2Suppressor of Cytokine Signaling 2細胞因子信號轉導抑制因子2調節細胞因子信號轉導的經典負反饋系統的一部分5.73×10?4
    Cpa1Carboxypeptidase A1羧肽酶A1催化C端氨基酸釋放,催化白三烯C4通過
    酰胺鍵的水解轉化為白三烯F4
    5.74×10?4
    Try10Trypsin 10胰蛋白酶10預測能夠激活絲氨酸型內肽酶活性,參與蛋白質水解7.94×10?4
    Try4Trypsin 4胰蛋白酶4能夠激活鈣離子結合活性和絲氨酸型內肽酶
    活性,參與蛋白質水解
    1.36×10?3
    SycnSyncollin微管成束蛋白在胰島細胞的外分泌中起作用,調節酶原顆粒的相互融合1.51×10?3
    CtrlChymotrypsin Like胰凝乳蛋白酶樣
    蛋白酶
    屬于肽酶S1家族,在胰腺中特異性表達,
    作為一種消化酶發揮作用
    1.84×10?3
    Guca2bGuanylate Cyclase Activator 2B鳥苷酸環化酶
    激活劑2B
    腸道鳥苷酸環化酶的內源性激活劑1.90×10?3
    Phgr1Proline, Histidine and Glycine Rich 1富含脯氨酸、組氨酸和甘氨酸1富含脯氨酸、組氨酸和甘氨酸2.01×10?3
    Prss2Serine Protease 2絲氨酸蛋白酶2屬于絲氨酸蛋白酶胰蛋白酶家族,編碼陰離子胰蛋白酶原2.48×10?3
    Ckmt1Creatine Kinase, Mitochondrial 1A肌酸激酶,線粒體1A可逆地催化磷酸在ATP和各種磷酸原
    (如磷酸肌酸)之間的轉移
    2.82×10?3
    Dmbt1Deleted In Malignant Brain Tumors 1惡性腦瘤缺失基因1在粘膜防御系統和細胞免疫防御中發揮作用3.62×10?3
    Ela3bChymotrypsin Like Elastase 3B胰凝乳蛋白酶樣彈性蛋白酶3具有丙氨酸特異性的高效蛋白酶,但僅有
    很弱的彈性蛋白分解活性
    3.68×10?3
    Try10lTry10-like trypsinogenTry10樣胰蛋白酶原胰蛋白酶原4.14×10?3
    Ms4a10Membrane Spanning 4-Domains A10跨膜4結構域A10可能作為多聚體受體復合物的一個組成部分參與信號轉導4.28×10?3
    CelCarboxyl Ester Lipase膽鹽活化脂肪酶促進脂肪和維生素的消化吸收4.41×10?3
    Eg436523Eg436523胰蛋白酶原12能夠激活絲氨酸型內肽酶活性,參與蛋白質水解,與人類PRSS3同源4.81×10?3
    Ai747448Ai747448鈣激活氯通道能夠激活細胞內鈣激活的氯離子通道活性,是質膜不可分割的組成部分,與人類CLCA4同源5.58×10?3
    Tyr5Tyrosinase 5酪氨酸酶5催化酪氨酸氧化為多巴醌,參與色素形成6.12×10?3
    Cpb1Carboxypeptidase B1羧肽酶B1參與肽類激素代謝和蛋白質代謝6.79×10?3
    ClraC-type lectin domain family 2,
    member e
    C型凝集素結構域家族2,成員e能激活自然殺傷細胞凝集素樣受體結合活性,與人類CLEC2D(C型凝集素結構域家族2成員D)同源7.02×10?3
    VdrVitamin D Receptor維生素D受體脂質代謝中的核受體,維生素D3的活性形式7.27×10?3
    Gal3st2Galactose-3-O-Sulfotransferase 2半乳糖-3-O-磺基
    轉移酶2
    將硫酸基團轉移到非還原β-半乳糖殘基的
    C-3位羥基上來催化磺化
    7.37×10?3
    Tm4sf5Transmembrane 4 L Six Family Member 5跨膜4 L六家族成員5在細胞增殖中起作用,其過度表達可能與
    腫瘤細胞不受控制的生長有關
    9.14×10?3
    KrtdapKeratinocyte Differentiation Associated Protein角質細胞分化
    相關蛋白
    角質細胞分化的可溶性調節劑,調節角質
    細胞分化和維持復層上皮
    9.00×10?5
    Myh8Myosin Heavy Chain 8肌球蛋白重鏈8在骨骼肌收縮中起作用2.08×10?4
    LorLoricrin Cornified Envelope Precursor Protein洛立克林角質化包膜前體蛋白參與角質化和發育生物學通路7.43×10?4
    Mzb1Marginal Zone B And B1 Cell Specific Protein邊緣區B和B1細胞特異性蛋白胰島素抵抗的發生中起作用,作為一種
    激素調節的脂肪因子/促炎細胞因子
    7.64×10?4
    Krt113Keratin 113角蛋白113屬于角蛋白基因家族9.22×10?4
    Myh4Myosin Heavy Chain 4肌球蛋白重鏈4與肌肉收縮相關2.25×10?3
    Myl3Myosin Light Chain 3肌球蛋白輕鏈3參與導向信號、生長錐運動和cAMP依賴性
    PKA的激活通路
    2.58×10?3
    PvalbParvalbumin副白蛋白與肌肉松弛有關3.85×10?3
    Ly6dLymphocyte Antigen 6 Family
    Member D
    淋巴細胞抗原6家族成員D參與翻譯后修飾GPI錨定蛋白的合成和
    蛋白質的代謝
    4.31×10?3
    RptnRepetin重復蛋白參與角質化細胞包膜的形成6.05×10?3
    Asprv1Aspartic Peptidase Retroviral Like 1天冬氨酸肽酶逆轉錄病毒樣1負責絲聚蛋白加工的蛋白酶,對維持
    正常表皮組織至關重要
    7.52×10?3
    Lce1cLate Cornified Envelope 1C晚期角質化外殼1C與角質化和發育生物學相關8.16×10?3
    Crct1Cysteine Rich C-Terminal 1富含半胱氨酸的C端1表達于左肺和右肺,與人CRCT1基因同源8.20×10?3
    Lgals7Galectin 7半乳糖凝集素7參與正常生長控制所必需的細胞-細胞和細胞-基質相互作用9.55×10?3
    下載: 導出CSV

    表  3  差異基因富集的信號通路

    Table  3.   Biological pathways of differential gene enrichment

    通路編號富集的通路基因P
    mmu04972Pancreatic secretionPnliprp1, Cpa1, Cpb1, Ctrl, Cel, Try10, Prss2, Pnlip, Try41.36×10-11
    mmu04974Protein digestion and absorptionCpa1, Cpb1, Ctrl, Try10, Prss2, Try49.71×10-7
    mmu04975Fat digestion and absorptionPnliprp1, Clps, Cel, Pnlip7.22×10-5
    mmu00561Glycerolipid metabolismPnliprp1, Cel, Pnlip7.00×10-3
    下載: 導出CSV

    表  4  關鍵節點基因的功能分析

    Table  4.   Functional analysis of key node genes

    序號基因名稱基因全稱基因功能
    1Pnliprp1胰脂肪酶相關蛋白1參與甘油三酯消化
    2Pnlip胰三酰甘油脂肪酶在脂肪代謝中起重要作用
    3Prss2絲氨酸蛋白酶2屬于絲氨酸蛋白酶胰蛋白酶家族,編碼陰離子胰蛋白酶原
    4Clps脂肪酶使脂肪酶將自身錨定在脂水界面上,發揮消化脂質功能
    5Tff2三葉因子2抑制胃腸蠕動和胃酸分泌
    6Ctrl胰凝乳蛋白酶樣蛋白酶屬于肽酶S1家族
    7Cpa1羧肽酶A1催化C端氨基酸的釋放
    8Cel膽鹽活化脂肪酶促進脂肪和維生素的消化吸收
    9Dmbt1惡性腦瘤缺失基因1在粘膜防御系統和細胞免疫防御中發揮作用
    10Vil1絨毛蛋白1肌動蛋白修飾蛋白,調節微絨毛肌動蛋白絲的重組
    下載: 導出CSV
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  • 收稿日期:  2021-11-10
  • 網絡出版日期:  2022-08-11
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