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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
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。
Establishment of a mouse model of vascular endothelial-mesenchymal transdifferentiation genetic tracing and its role in liver fibrosis studies
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摘要:
目的 构建Cdh5-CreERT/Acta2-tdTomato-STOPfloxed-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-CreERT小鼠和Acta2-KI小鼠进行交配繁育, 经PCR基因型鉴定得到Cdh5-CreERT/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-CreERT/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-CreERT/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。 Abstract:Objective To establish Cdh5-CreERT/Acta2-tdTomato-STOPfloxed-eGFP knockin genetic tracing mice, and to investigate its application in studies on vascular endothelial cell transition in liver fibrosis. Methods Cdh5-CreERT mice were mated with Acta2-KI mice, and the Cdh5-CreERT/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-CreERT/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-CreERT/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. -
Key words:
- Liver Cirrhosis /
- Epithelial-Mesenchymal Transition /
- Endothelial Cells /
- Mice
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图 1 Cdh5-CreERT/Acta2小鼠的构建策略与作用机制
Figure 1. Establishing strategy and mechanism of Cdh5-CreERT/Acta2 mice
注：(A)为Cdh5-CreERT条带；(B) 为Acta2-KI突变型条带；(C) 为Acta2-KI野生型条带。
图 2 子代小鼠基因组PCR鉴定结果
Figure 2. PCR results of offspring mice's genomic identification
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
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
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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