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

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

张杰¹,
马学峰¹,
王艺奋¹,
王孟轲¹,
庄立琨²,
刘守胜²,
辛永宁^{1, 3, ,}

1.
青岛大学附属青岛市市立医院感染性疾病科，山东青岛 266011
2.
青岛大学附属青岛市市立医院临床研究中心，山东青岛 266071
3.
青岛市消化疾病重点实验室，山东青岛 266071

基金项目:

国家自然科学基金 (31770837)

详细信息

通信作者:
辛永宁，xinyongning@163.com

中图分类号: R575.5
计量
- 文章访问数: 1256
- HTML全文浏览量: 359
- PDF下载量: 104
- 被引次数: 0
出版历程
- 收稿日期: 2021-04-26
- 录用日期: 2021-06-04
- 出版日期: 2021-11-20

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

1.
Department of Infectious Diseases, Qingdao Municipal Hospital, Qingdao University, Qingdao, Shandong 266011, China
2.
Clinical Research Center, Qingdao Municipal Hospital, Qingdao University, Qingdao, Shandong 266071, China
3.
Qingdao Key Laboratory of Digestive Diseases, Qingdao, Shandong 266071, China

Research funding:

National Natural Science Foundation of China (31770837)

摘要

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。
- 非酒精性脂肪性肝病 /
- TM6SF2 /
- CRISPR/Cas9
Abstract: Objective To establish a mouse model of hepatocyte-specific TM6SF2 knockout, and to investigate the role of TM6SF2 in the development of nonalcoholic fatty liver disease (NAFLD). Methods The CRISPR/Cas9 technique and the Cre/LoxP strategy were used to establish a stable mouse model of hepatocyte-specific TM6SF2 knockout. The mice with hepatocyte-specific TM6SF2 knockout and the control mice were given a normal diet or a high-fat diet (HFD) for 16 weeks, and related indices were measured, including general status (body weight and liver weight), glucose metabolic indices (fasting blood glucose and insulin), and lipid metabolism (plasma triglyceride, cholesterol, and liver triglyceride). The t-test was used for comparison of normally distributed continuous data between two groups. Results Under the condition of HFD, compared with the control mice, the mice with hepatocyte-specific TM6SF2 knockout had significantly higher liver weight (2.235±0.175 g vs 1.258±0.106 g, t=4.789, P < 0.01) and liver index (4.970%±0.298% vs 3.210%±0.094%, t=5.630, P < 0.01), and the loss of the TM6SF2 gene in hepatocytes aggravated the abnormal level of alanine aminotransferase induced by HFD (62.517±1.526 U/L vs 25.991±5.947 U/L, t=5.949, P < 0.01). Compared with the control mice under the condition of normal diet or HFD, the mice with TM6SF2 knockout had a significant increase in plasma insulin level (normal diet: 37.203±0.836 mIU/L vs 34.835±0.426 mIU/L, t=2.520, P=0.025; HFD: 41.093±1.226 mIU/L vs 35.817±0.500 mIU/L, t=3.985, P=0.007), while there were no significant differences in the other indices associated with glucose metabolism (all P > 0.05). Under the condition of HFD, there were no significant differences in the levels of plasma triglyceride and cholesterol between the mice with hepatocyte-specific TM6SF2 knockout and the control group (P > 0.05), while the mice with hepatocyte-specific TM6SF2 knockout had a significant increase in the level of liver triglyceride compared with the control mice (23.969±0.978 mg/g vs 18.229±1.633 mg/g, t=3.015, P=0.024). Conclusion Hepatocyte-specific knockout of TM6SF2 can aggravate liver lipid accumulation and liver injury in mice with NAFLD.
- Non-alcoholic Fatty Liver Disease /
- TM6SF2 /
- CRISPR/Cas9

HTML全文

图 1 TM6SF2-CKO小鼠构建策略

下载: 全尺寸图片幻灯片

图 2 TM6SF2-HKO小鼠基因型鉴定

注：a, TM6SF2^fl/fl鉴定, 电泳仅有一条332 bp条带，则为TM6SF2^fl/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TM6SF2^fl/-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(TM6SF2^fl/fl Alb-Cre)，45号为TM6SF2-Flox小鼠(TM6SF2^fl/fl)。

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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。

下载: 全尺寸图片幻灯片

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。

下载: 全尺寸图片幻灯片

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值	P值	TM6SF2-Flox- HFD(n=4)	TM6SF2-HKO- HFD (n=4)	t值	P值
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值	P值	TM6SF2-Flox- HFD(n=4)	TM6SF2-HKO- HFD(n=4)	t值	P值
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值	TM6SF2-Flox- HFD(n=4)	TM6SF2-HKO- HFD(n=4)	t值	P值
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

参考文献(19)

[1]	COBBINA E, AKHLAGHI F. Non-alcoholic fatty liver disease (NAFLD) - pathogenesis, classification, and effect on drug metabolizing enzymes and transporters[J]. Drug Metab Rev, 2017, 49(2): 197-211. DOI: 10.1080/03602532.2017.1293683.
[2]	WU TF, LIAO XH, ZHONG BH. Epidemiology of nonalcoholic fatty liver disease in some regions of China[J]. J Clin Hepatol, 2020, 36(6): 1370-1373. DOI: 10.3969/j.issn.1001-5256.2020.06.039. 吴挺丰, 廖献花, 钟碧慧. 中国部分地区非酒精性脂肪肝病的流行情况[J]. 临床肝胆病杂志, 2020, 36(6): 1370-1373. DOI: 10.3969/j.issn.1001-5256.2020.06.039.
[3]	YOUNOSSI Z, ANSTEE QM, MARIETTI M, et al. Global burden of NAFLD and NASH: Trends, predictions, risk factors and prevention[J]. Nat Rev Gastroenterol Hepatol, 2018, 15(1): 11-20. DOI: 10.1038/nrgastro.2017.109.
[4]	BYRNE CD, TARGHER G. EASL-EASD-EASO clinical practice guidelines for the management of non-alcoholic fatty liver disease: Is universal screening appropriate?[J]. Diabetologia, 2016, 59(6): 1141-1144. DOI: 10.1007/s00125-016-3910-y.
[5]	LEUNG C, RIVERA L, FURNESS JB, et al. The role of the gut microbiota in NAFLD[J]. Nat Rev Gastroenterol Hepatol, 2016, 13(7): 412-425. DOI: 10.1038/nrgastro.2016.85.
[6]	KOZLITINA J, SMAGRIS E, STENDER S, et al. Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease[J]. Nat Genet, 2014, 46(4): 352-356. DOI: 10.1038/ng.2901.
[7]	SOOKOIAN S, CASTAÑO GO, SCIAN R, et al. Genetic variation in transmembrane 6 superfamily member 2 and the risk of nonalcoholic fatty liver disease and histological disease severity[J]. Hepatology, 2015, 61(2): 515-525. DOI: 10.1002/hep.27556.
[8]	KRAWCZYK M, RAU M, SCHATTENBERG JM, et al. Combined effects of the PNPLA3 rs738409, TM6SF2 rs58542926, and MBOAT7 rs641738 variants on NAFLD severity: A multicenter biopsy-based study[J]. J Lipid Res, 2017, 58(1): 247-255. DOI: 10.1194/jlr.P067454.
[9]	GOFFREDO M, CAPRIO S, FELDSTEIN AE, et al. Role of TM6SF2 rs58542926 in the pathogenesis of nonalcoholic pediatric fatty liver disease: A multiethnic study[J]. Hepatology, 2016, 63(1): 117-125. DOI: 10.1002/hep.28283.
[10]	ANDRIKOPOULOS S, BLAIR AR, DELUCA N, et al. Evaluating the glucose tolerance test in mice[J]. Am J Physiol Endocrinol Metab, 2008, 295(6): E1323-1332. DOI: 10.1152/ajpendo.90617.2008.
[11]	MA Y, YU L, PAN S, et al. CRISPR/Cas9-mediated targeting of the Rosa26 locus produces Cre reporter rat strains for monitoring Cre-loxP-mediated lineage tracing[J]. FEBS J, 2017, 284(19): 3262-3277. DOI: 10.1111/febs.14188.
[12]	FAN Y, LU H, GUO Y, et al. Hepatic Transmembrane 6 superfamily member 2 regulates cholesterol metabolism in mice[J]. Gastroenterology, 2016, 150(5): 1208-1218. DOI: 10.1053/j.gastro.2016.01.005.
[13]	GUPTA D, BHATTACHARJEE O, MANDAL D, et al. CRISPR-Cas9 system: A new-fangled dawn in gene editing[J]. Life Sci, 2019, 232: 116636. DOI: 10.1016/j.lfs.2019.116636.
[14]	HRYHOROWICZ M, LIPIŃSKI D, ZEYLAND J, et al. CRISPR/Cas9 immune system as a tool for genome engineering[J]. Arch Immunol Ther Exp (Warsz), 2017, 65(3): 233-240. DOI: 10.1007/s00005-016-0427-5.
[15]	YANG J, ZHU D, JU B, et al. Hepatoprotective effects of Gentianella turkestanerum extracts on acute liver injury induced by carbon tetrachloride in mice[J]. Am J Transl Res, 2017, 9(2): 569-579.
[16]	HWANG YP, CHOI JH, JEONG HG. Protective effect of the Aralia continentalis root extract against carbon tetrachloride-induced hepatotoxicity in mice[J]. Food Chem Toxicol, 2009, 47(1): 75-81. DOI: 10.1016/j.fct.2008.10.011.
[17]	Diagnosis and Treatment Center of Hepatology of South China Alliance of TCM, National Administration of Traditional Chinese Medicine. Diagnosis and treatment scheme of Ganpi (non-alcoholic steatohepatitis)[J/CD]. Chin J Liver Dis: Electronic Edition, 2021, 13(1): 1-9. DOI: 10.3969/j.issn.1674-7380.2021.01.001 国家中医药管理局华南区中医肝病诊疗中心联盟. 肝癖(非酒精性脂肪性肝炎)诊疗方案[J/CD]. 中国肝脏病杂志(电子版), 2021, 13(1): 1-9. DOI: 10.3969/j.issn.1674-7380.2021.01.001
[18]	PETERSEN MC, VATNER DF, SHULMAN GI. Regulation of hepatic glucose metabolism in health and disease[J]. Nat Rev Endocrinol, 2017, 13(10): 572-587. DOI: 10.1038/nrendo.2017.80.
[19]	WATT MJ, MIOTTO PM, de NARDO W, et al. The liver as an endocrine organ-linking nafld and insulin resistance[J]. Endocr Rev, 2019, 40(5): 1367-1393. DOI: 10.1210/er.2019-00034.