血管内皮间质样转分化遗传示踪小鼠模型的构建及其在肝纤维化研究中的应用

许皓; 阮柏; 历志文; 房志强; 王琳; 窦科峰

doi:10.3969/j.issn.1001-5256.2022.04.018

血管内皮间质样转分化遗传示踪小鼠模型的构建及其在肝纤维化研究中的应用

DOI: 10.3969/j.issn.1001-5256.2022.04.018

许皓¹,
阮柏^{1, 2},
历志文¹,
房志强¹,
王琳¹,
窦科峰^1, , ,

1.
空军军医大学西京医院肝胆胰脾外科, 西安 710032
2.
空军军医大学航空航天医学系航空航天临床医学中心, 西安 710032

基金项目:

国家重点研发计划项目 (2016YFA0102100);

国家自然科学基金 (81770560);

国家自然科学基金 (81800533)

伦理学声明：动物实验操作符合空军军医大学西京医院伦理委员会标准，伦理审批时间: 2018年3月6日，批号：KY20183238-1。

利益冲突声明：本研究不存在研究者、伦理委员会成员以及与公开研究成果有关的利益冲突。

作者贡献声明：许皓、阮柏完成实验设计，分析数据，撰写文章；历志文、房志强协助实验操作和数据收集分析；王琳、窦科峰指导研究思路，修改文章并定稿。

详细信息

通信作者:
窦科峰, doukef@fmmu.edu.cn

许皓与阮柏对本文贡献等同，同为第一作者

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出版历程
- 收稿日期: 2021-10-26
- 录用日期: 2021-11-28
- 出版日期: 2022-04-20

Establishment of a mouse model of vascular endothelial-mesenchymal transdifferentiation genetic tracing and its role in liver fibrosis studies

Hao XU¹,
Bai RUAN^{1, 2},
Zhiwen LI¹,
Zhiqiang FANG¹,
Lin WANG¹,
Kefeng DOU^{1
, ,
,}

1.
Department of Hepatobiliary, Pancreatic and Splenic Surgery, Xijing Hospital, Air Force Medical University, Xi'an 710032, China
2.
Center of Clinical Aerospace Medicine, School of Aerospace Medicine, Air Force Medical University, Xi'an 710032, China

Research funding:

The National Key Research and Development Program of China (2016YFA0102100);

National Natural Science Foundation of China (81770560);

National Natural Science Foundation of China (81800533)

More Information

Corresponding author: DOU Kefeng, doukef@fmmu.edu.cn (ORCID: 0000-0002-8857-5663)

摘要

摘要: 目的构建Cdh5-Cre^ERT/Acta2-tdTomato-STOP^floxed-eGFP knockin遗传示踪小鼠, 并探究其在肝纤维化血管内皮细胞转化研究中的应用。方法将Cdh5-Cre^ERT小鼠和Acta2-KI小鼠进行交配繁育, 经PCR基因型鉴定得到Cdh5-Cre^ERT/Acta2-KI遗传示踪小鼠。通过分离、培养原代肝血窦内皮细胞(LSEC)和建立CCl₄肝纤维化模型, 取LSEC和肝脏组织, 分别进行免疫荧光染色, 观察荧光蛋白tdTomato和eGFP的表达。结果经他莫昔芬诱导后, Cdh5-Cre^ERT/Acta2-KI遗传示踪小鼠的LSEC和肝脏组织在体内外建立的内皮-间质样转分化条件下可表达eGFP, 而未经诱导的对照组只表达tdTomato。结论成功构建的Cdh5-Cre^ERT/Acta2-KI遗传示踪小鼠可实现对血管内皮-间质样转分化的有效标记, 并为肝纤维化中肌纤维母细胞来源的多样性提供新的遗传示踪学依据。
- 肝硬化 /
- 上皮-间质转化 /
- 内皮细胞 /
- 小鼠
Abstract: Objective To establish Cdh5-Cre^ERT/Acta2-tdTomato-STOP^floxed-eGFP knockin genetic tracing mice, and to investigate its application in studies on vascular endothelial cell transition in liver fibrosis. Methods Cdh5-Cre^ERT mice were mated with Acta2-KI mice, and the Cdh5-Cre^ERT/Acta2-KI genetic tracing mice were obtained and identified by PCR genotyping. Primary liver sinusoid endothelial cells (LSECs) were isolated and cultured, and a model of CCl₄-induced liver fibrosis was established. LSECs and liver tissue were collected for immunofluorescent staining to observe the expression of the fluorescent proteins tdTomato and eGFP. Results After being induced by tamoxifen, LSECs and liver tissue of Cdh5-Cre^ERT/Acta2-KI genetic tracing mice expressed eGFP under the conditions for epithelial-mesenchymal transdifferentiation established in vivo and in vitro, while the control group without induction expressed tdTomato alone. Conclusion The successfully established Cdh5-Cre^ERT/Acta2-KI genetic tracing mice can realize the effective labeling of epithelial-mesenchymal transdifferentiation, which provides a genetic tracing basis for the diverse sources of mesenchymal myofibroblasts in liver fibrosis.
- Liver Cirrhosis /
- Epithelial-Mesenchymal Transition /
- Endothelial Cells /
- Mice

HTML全文

图 1 Cdh5-Cre^ERT/Acta2小鼠的构建策略与作用机制

Figure 1. Establishing strategy and mechanism of Cdh5-Cre^ERT/Acta2 mice

下载: 全尺寸图片幻灯片

图 2 子代小鼠基因组PCR鉴定结果

注：(A)为Cdh5-Cre^ERT条带；(B) 为Acta2-KI突变型条带；(C) 为Acta2-KI野生型条带。

Figure 2. PCR results of offspring mice's genomic identification

下载: 全尺寸图片幻灯片

图 3 体外培养7 d后LSEC和HSC的免疫荧光染色

Figure 3. Immunofluorescent staining of LSEC and HSC after 7 d culture in vitro

下载: 全尺寸图片幻灯片

图 4 CCl₄诱导肝纤维化模型肝脏组织免疫荧光染色

Figure 4. Immunofluorescent staining of liver tissue for CCl₄ induced fibrotic model

下载: 全尺寸图片幻灯片

表 1 PCR扩增引物

Table 1. PCR amplification primer

引物名称	引物序列(5′-3′)	引物类别
Cdh5 N1	CCGGTCGATGCAACGAGTGATGAGG	正向引物
Cdh5 N2	GCCTCCAGCTTGCATGATCTCCGG	反向引物
Acta2 5F1	AAAGCTGATGCTTGCCACTTC	突变型正向引物
Acta2 5R1	GATGACGGCCATGTTGTTGTC	突变型反向引物
Acta2 wtF3	CAGCTATGTGTGAAGAGGAAGACAG	野生型正向引物
Acta2 wtR3	GCACCTTGAACCACTAGGTTATATCC	野生型反向引物

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参考文献(15)

[1]	LEE UE, FRIEDMAN SL. Mechanisms of hepatic fibrogenesis[J]. Best Pract Res Clin Gastroenterol, 2011, 25(2): 195-206. DOI: 10.1016/j.bpg.2011.02.005.
[2]	MEDERACKE I, HSU CC, TROEGER JS, et al. Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology[J]. Nat Commun, 2013, 4: 2823. DOI: 10.1038/ncomms3823.
[3]	RAY K. Liver: hepatic stellate cells hold the key to liver fibrosis[J]. Nat Rev Gastroenterol Hepatol, 2014, 11(2): 74. DOI: 10.1038/nrgastro.2013.244.
[4]	PIERA-VELAZQUEZ S, MENDOZA FA, JIMENEZ SA. Endothelial to mesenchymal transition (EndoMT) in the pathogenesis of human fibrotic diseases[J]. J Clin Med, 2016, 5(4): 45. DOI: 10.3390/jcm5040045.
[5]	SUN X, NKENNOR B, MASTIKHINA O, et al. Endothelium-mediated contributions to fibrosis[J]. Semin Cell Dev Biol, 2020, 101: 78-86. DOI: 10.1016/j.semcdb.2019.10.015.
[6]	RUAN B, DUAN JL, XU H, et al. Capillarized liver sinusoidal endothelial cells undergo partial endothelial-mesenchymal transition to actively deposit sinusoidal ECM in liver fibrosis[J]. Front Cell Dev Biol, 2021, 9: 671081. DOI: 10.3389/fcell.2021.671081.
[7]	THOMSON JG, RUCKER EB 3rd, PIEDRAHITA JA. Mutational analysis of loxP sites for efficient Cre-mediated insertion into genomic DNA[J]. Genesis, 2003, 36(3): 162-167. DOI: 10.1002/gene.10211.
[8]	KOS CH. Cre/loxP system for generating tissue-specific knockout mouse models[J]. Nutr Rev, 2004, 62(6 Pt 1): 243-246. DOI: 10.1301/nr2004.jun243-246.
[9]	JIANG ZJ, YUE ZS, YANG YC, et al. Improvement method of isolation of mouse liver Sinusoidal endothelial cell[J]. Prog Mod Biomed, 2018, 18(6): 1034-1039. DOI: 10.13241/j.cnki.pmb.2018.06.007. 蒋子剑, 岳振生, 杨毅聪, 等. 小鼠肝血窦内皮细胞分离与鉴定新方法[J]. 现代生物医学进展, 2018, 18(6): 1034-1039. DOI: 10.13241/j.cnki.pmb.2018.06.007.
[10]	WANG S, FRIEDMAN SL. Hepatic fibrosis: A convergent response to liver injury that is reversible[J]. J Hepatol, 2020, 73(1): 210-211. DOI: 10.1016/j.jhep.2020.03.011.
[11]	TERKELSEN MK, BENDIXEN SM, HANSEN D, et al. Transcriptional dynamics of hepatic sinusoid-associated cells after liver injury[J]. Hepatology, 2020, 72(6): 2119-2133. DOI: 10.1002/hep.31215.
[12]	TRAUTWEIN C, FRIEDMAN SL, SCHUPPAN D, et al. Hepatic fibrosis: Concept to treatment[J]. J Hepatol, 2015, 62(1 Suppl): S15-S24. DOI: 10.1016/j.jhep.2015.02.039.
[13]	ZEISBERG EM, TARNAVSKI O, ZEISBERG M, et al. Endothelial-to-mesenchymal transition contributes to cardiac fibrosis[J]. Nat Med, 2007, 13(8): 952-961. DOI: 10.1038/nm1613.
[14]	ZEISBERG EM, POTENTA SE, SUGIMOTO H, et al. Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition[J]. J Am Soc Nephrol, 2008, 19(12): 2282-2287. DOI: 10.1681/ASN.2008050513.
[15]	HASHIMOTO N, PHAN SH, IMAIZUMI K, et al. Endothelial-mesenchymal transition in bleomycin-induced pulmonary fibrosis[J]. Am J Respir Cell Mol Biol, 2010, 43(2): 161-172. DOI: 10.1165/rcmb.2009-0031OC.