Nature Communications | 谱系示踪技术揭示肝脏损伤和再生的秘密

2016-11-22 12:37 by 南模生物


肝细胞在功能上具有异质性的特点,根据代谢区带化可以将肝细胞分成两个不同的群体:门静脉周围的肝细胞和中心周围的肝细胞。


中科院上海生命科学研究院营养科学研究所周斌课题组的研究论文揭示了在肝脏损伤和再生过程中,这两类细胞的细胞动力学过程。该成果发表于2016年11月18日的Nature Communications杂志上,题为“Mfsd2a+ hepatocytes repopulate the liver during injury and regeneration”。


维持血脑屏障功能的重要成员Mfsd2a(major facilitator super family domain containing 2a)被发现是门静脉周围肝细胞的标志Marker。在Mfsd2a基因起始密码子位置插入CreER诱导型重组酶,建立Mfsd2a-CreER基因敲入小鼠。再与Rosa26-LSL-RFP荧光报告基因小鼠交配,对Mfsd2a阳性门静脉周围肝细胞进行谱系示踪。研究人员发现Mfsd2a阳性细胞群在肝脏内稳态过程中逐渐减少。而部分肝切除后的肝再生过程会显著促进Mfsd2a阳性门静脉周围肝细胞的增殖。与之相似的是,在慢性肝损伤过程中,Mfsd2a阳性肝细胞群也会扩张,并完全取代中心周围肝细胞群。肝损伤恢复之后,Mfsd2a阳性肝细胞被重编程变为中心周围肝细胞,肝脏代谢区带被重新建立起来。

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Fig1. Schematic figures showing (a) our knock-in strategy for Mfsd2a-CreER allele using CRISPR/Cas9 by homologous recombination and (b) genetic lineage tracing strategy for Mfsd2a+ hepatocytes by Cre-LoxP recombination in Mfsd2a+ hepatocytes. (c) Whole-mount fluorescence view of the adult liver from 6-week-old Mfsd2a-CreER;Rosa26-RFP mice. Tamoxifen was induced at 2 days before analysis. Scale bar, 1 mm. (d) Immunostaining for RFP, CK19 and HNF4a on liver sections shows Mfsd2a-expressing hepatocytes in PP zone (P). Scale bars, 100 μm. (e) Immunostaining for RFP, PECAM and 4,6-diamidino-2-phenylindole (DAPI) on liver sections shows Mfsd2a-expressing hepatocytes in the PP zone but not PC zone (*). Scale bars, 100 μm. (f) Isolation of RFP− and RFP+ cells by flow cytometry followed by quantitative RT–PCR (qRT–PCR) analysis for expression of RFP and Mfsd2a. Expression level of genes in the RFP− cells was set as 1. Error bars are s.e.m. of the mean for all the quantification in this study. (g) Expression of PP and PC genes detected by qRT–PCR. The x axis denotes RFP− (black) and RFP+ (red) groups, and the y axis denotes fold induction.. *P<0.05; n=3, two-tailed unpaired t-test. Each immunostaining image is a representative of four individual samples.


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