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肝脏TM6SF2特异性敲除促进非酒精性脂肪性肝病小鼠肝脏脂肪变性

张杰 马学峰 王艺奋 王孟轲 庄立琨 刘守胜 辛永宁

张杰,马学峰,王艺奋,等. 肝脏TM6SF2特异性敲除促进非酒精性脂肪性肝病小鼠肝脏脂肪变性[J]. 临床肝胆病杂志, 2021, 37(11): 2612-2616. DOI: 10.3969/j.issn.1001-5256.2021.11.024
引用本文: 张杰,马学峰,王艺奋,等. 肝脏TM6SF2特异性敲除促进非酒精性脂肪性肝病小鼠肝脏脂肪变性[J]. 临床肝胆病杂志, 2021, 37(11): 2612-2616. DOI: 10.3969/j.issn.1001-5256.2021.11.024
ZHANG J, MA XF, WANG YF, et al. Hepatocyte-specific TM6SF2 knockout aggravates hepatic steatosis in mice with nonalcoholic fatty liver disease[J]. J Clin Hepatol, 2021, 37(11): 2612-2616. DOI: 10.3969/j.issn.1001-5256.2021.11.024
Citation: ZHANG J, MA XF, WANG YF, et al. Hepatocyte-specific TM6SF2 knockout aggravates hepatic steatosis in mice with nonalcoholic fatty liver disease[J]. J Clin Hepatol, 2021, 37(11): 2612-2616. DOI: 10.3969/j.issn.1001-5256.2021.11.024

肝脏TM6SF2特异性敲除促进非酒精性脂肪性肝病小鼠肝脏脂肪变性

DOI: 10.3969/j.issn.1001-5256.2021.11.024
基金项目: 

国家自然科学基金 31770837

详细信息
    通讯作者:

    辛永宁,xinyongning@163.com

  • 中图分类号: R575.5

Hepatocyte-specific TM6SF2 knockout aggravates hepatic steatosis in mice with nonalcoholic fatty liver disease

Research funding: 

National Natural Science Foundation of China 31770837

  • 摘要:   目的  构建TM6SF2肝细胞特异性敲除的小鼠模型,研究TM6SF2在非酒精性脂肪性肝病(NAFLD)发病中的作用。  方法  利用CRISPR/Cas9技术和Cre/LoxP策略构建稳定的TM6SF2肝细胞特异性敲除的小鼠模型。给予TM6SF2肝细胞特异性敲除小鼠和对照小鼠普通饮食或高脂饮食喂养16周,检测小鼠体质量、肝质量等一般情况,空腹血糖、胰岛素等糖代谢相关指标,以及血浆甘油三酯、胆固醇、肝脏甘油三酯等脂代谢相关的指标。符合正态分布的计量资料两组之间比较使用t检验。  结果  在高脂饮食条件下,TM6SF2肝细胞特异性敲除小鼠的肝质量[(2.235±0.175)g vs (1.258±0.106)g,t=4.789, P<0.01]和肝指数(4.970%± 0.298% vs 3.210%±0.094%,t=5.630, P<0.01)明显高于对照小鼠。同时,肝细胞TM6SF2基因缺失也会加重高脂饮食诱导的ALT水平异常[(62.517±1.526)U/L vs (25.991±5.947)U/L, t=5.949, P<0.01]。相较于对照组,在普通饮食和高脂饮食条件下,TM6SF2敲除小鼠血浆胰岛素水平升高[(37.203±0.836)mIU/L vs(34.835±0.426)mIU/L,t=2.520, P=0.025;(41.093±1.226)mIU/L vs(35.817 ±0.500)mIU/L,t=3.985, P=0.007],其他与糖代谢相关指标差异均无统计学意义(P值均>0.05)。在高脂饮食条件下,TM6SF2肝细胞特异性敲除小鼠血浆甘油三酯和胆固醇水平较对照组差异无统计学意义(P值均>0.05),而肝脏内甘油三酯水平较对照组升高[(23.969±0.978)mg/g vs(18.229±1.633) mg/g,t=3.015, P=0.024]。  结论  肝细胞特异性敲除TM6SF2可加重NAFLD小鼠肝脏脂质蓄积和肝损伤。

     

  • 图  1  TM6SF2-CKO小鼠构建策略

    图  2  TM6SF2-HKO小鼠基因型鉴定

    注:a, TM6SF2fl/fl鉴定, 电泳仅有一条332 bp条带,则为TM6SF2fl/fl纯合小鼠;仅有一条239 bp条带,则为TM6SF2-/-野生小鼠;若两条带都存在,则为TM6SF2fl/-杂合小鼠。b, Alb-Cre鉴定,若有一条279 bp条带,则为Cre转基小鼠。因此,42号为TM6SF2-HKO小鼠(TM6SF2fl/fl Alb-Cre),45号为TM6SF2-Flox小鼠(TM6SF2fl/fl)。

    图  3  TM6SF2-HKO和TM6SF2-Flox小鼠各组织TM6SF2表达水平

    图  4  四组小鼠肝脏HE染色(×200)

    注:a,TM6SF2-Flox-CD;b,TM6SF2-HKO-CD;c,TM6SF2-Flox-HFD;d,TM6SF2-HKO-HFD。

    图  5  四组小鼠油红O染色(×200)

    注:a,TM6SF2-Flox-CD;b,TM6SF2-HKO-CD;c,TM6SF2-Flox-HFD;d,TM6SF2-HKO-HFD。

    表  1  基因型鉴定对应的引物序列

    引物 序列(5′→3′)
    TM6SF2-F CAGTGTCAAGATTAGTGTTGGC
    TM6SF2-R CACCGAAGTTATACAGTCTGAT
    Cre-F ATATCTCACGTACTGACGGTGG
    Cre-R CCTTAGCGCCGTAAATCAATC
    下载: 导出CSV

    表  2  TM6SF2-HKO对小鼠肝质量、肝酶等指标影响

    指标 TM6SF2-Flox-CD(n=8) TM6SF2-HKO-CD(n=8) t P TM6SF2-Flox- HFD(n=4) TM6SF2-HKO- HFD (n=4) t P
    体质量(g) 25.996±0.483 27.150±0.545 1.585 0.135 39.088±2.436 44.830±0.937 2.200 0.070
    肝质量(g) 1.115±0.032 1.117±0.027 0.417 0.967 1.258±0.106 2.235±0.175 4.789 0.003
    肝指数(%) 4.284±0.076 4.111±0.050 1.900 0.078 3.210±0.094 4.970±0.298 5.630 0.001
    血浆ALT (U/L) 15.014±2.670 10.377±1.433 1.530 0.148 25.991±5.947 62.517±1.526 5.949 0.001
    血浆AST (U/L) 30.343±1.723 26.525±1.649 1.601 0.132 26.645±2.576 33.485±3.075 1.705 0.139
    注:CD,普通饮食;HFD,高脂饮食。
    下载: 导出CSV

    表  3  肝细胞TM6SF2缺失对小鼠糖代谢影响

    指标 TM6SF2-Flox- CD(n=8) TM6SF2-HKO- CD(n=8) t P TM6SF2-Flox- HFD(n=4) TM6SF2-HKO- HFD(n=4) t P
    空腹血糖(mmol/L) 7.495±0.571 7.347±0.403 0.211 0.836 9.877±1.017 9.489±0.621 0.325 0.756
    胰岛素(mIU/L) 34.835±0.426 37.203±0.836 2.520 0.025 35.817±0.500 41.093±1.226 3.985 0.007
    OGTT-0 min-血糖(mmol/L) 6.425±0.373 5.875±0.653 0.731 0.477 9.600±0.402 10.000±1.042 0.358 0.732
    OGTT-30 min-血糖(mmol/L) 15.200±0.645 14.980±0.666 0.243 0.812 21.830±2.862 24.38±1.518 0.787 0.461
    OGTT-60 min-血糖(mmol/L) 9.275±0.609 9.263±0.368 0.018 0.986 22.080±3.726 20.050±1.599 0.500 0.635
    OGTT-120 min-血糖(mmol/L) 5.900±0.403 6.438±0.455 0.884 0.392 15.080±1.805 12.630±1.278 1.108 0.310
    OGTT曲线下面积(mol·min/L) 1.147±0.043 1.147±0.053 0.008 0.994 2.162±0.141 2.244±0.308 0.243 0.817
    胰岛素抵抗指数(mmol·mIU/L2) 11.608±0.921 12.134±0.689 0.458 0.654 15.694±1.528 17.332±1.227 0.836 0.435
    下载: 导出CSV

    表  4  TM6SF2-HKO对小鼠脂代谢影响

    指标 TM6SF2-Flox- CD(n=8) TM6SF2-HKO- CD(n=8) t值 P TM6SF2-Flox- HFD(n=4) TM6SF2-HKO- HFD(n=4) t P
    血浆TC(mmol/L) 1.182±0.235 1.159±0.191 0.772 0.940 2.203±0.661 2.337±0.681 0.940 0.383
    血浆TG(mmol/L) 0.874±0.048 0.724±0.058 2.003 0.065 1.046±0.213 0.914±0.010 0.563 0.594
    肝组织TG(mg/g) 11.935±0.597 13.240±0.658 1.467 0.164 18.229±1.633 23.969±0.978 3.015 0.024
    下载: 导出CSV
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  • 收稿日期:  2021-04-26
  • 录用日期:  2021-06-04
  • 出版日期:  2021-11-20
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