Gene Expression in Perivenous Hepatocytes: A Laser Capture Microdissection and Transcriptomic Analysis.

Differential expression of genes and proteins, as well as physiological processes, along the hepatic acinus, known collectively as hepatic zonation, is a fundamental feature of hepatic physiology. However, the precise cellular location of only a relatively small proportion of the ~14,000 genes expressed in the liver is known. This is critical information for understanding hepatic physiology as well as for the accurate targeting of gene transfer vectors for gene therapy. We have employed laser capture microdissection (LCM) and unbiased transcriptomic analysis of the normal adult mouse liver to define gene expression in a very distinct population of cells in the acinus, the glutamine-synthetase sub-compartment of perivenous hepatocytes, encompassing a one- to two-layer of cells surrounding the centrilobular vein. We confirmed the location of a number of genes whose expression is known to be restricted to these cells by virtue of immunohistochemistry or in situ hybridization (Slc1a2, Glul, Rhbg). We also identified genes whose expression had not previously been reported to be enriched in these cells (e.g. Sp5, Vldlr, Lpl, Gabrb3, Rcan2). Transcription factor analysis of the differentially expressed genes suggested important roles, in these cells, for members of the polycomb group, Wt1 and Tbx3. Collectively, our findings highlight the utility of combined LCM and transcriptome analysis for the identification of novel functions of distinct subclasses of hepatocytes. We plan to extend this approach to the mapping of physiological function across hepatic acini of the human liver. Comparison of mRNA expression by two hepatic cell populations: the glutamine synthetase-subcompartment of the perivenous compartment and zones 1+2 of the hepatic acinus Cells were isolated by laser capture microdissection and mRNA extracted by standard protocol.

基因与蛋白质的差异表达，以及沿肝腺泡分布的生理过程，统称为肝分区（hepatic zonation），是肝脏生理学的核心特征之一。然而，目前仅明确了肝脏表达的约14000个基因中，相对较小一部分的精准细胞定位。这一信息对于解析肝脏生理学，以及实现基因治疗中基因转移载体的精准靶向均至关重要。本研究通过激光捕获显微切割（laser capture microdissection, LCM）技术，结合正常成年小鼠肝脏的无偏转录组分析，明确了肝腺泡中一类极为独特的细胞群——静脉周肝细胞的谷氨酰胺合成酶亚区——的基因表达特征，该亚区包含围绕中央静脉的1~2层细胞。我们通过免疫组织化学或原位杂交技术，验证了一批已知仅在该细胞群中表达的基因的定位（如Slc1a2、Glul、Rhbg）。此外，我们还发现了一批此前未见报道在该细胞群中富集表达的基因（如Sp5、Vldlr、Lpl、Gabrb3、Rcan2）。对差异表达基因的转录因子分析显示，多梳蛋白家族成员、Wt1及Tbx3在该细胞群中发挥重要调控作用。综上，本研究结果证实了联合应用LCM与转录组分析技术，可用于发掘肝细胞不同亚群的全新功能。我们计划将该方法推广应用于人肝肝腺泡的生理功能图谱绘制。本研究对两类肝细胞群的mRNA表达水平进行了比较：一是静脉周区域的谷氨酰胺合成酶亚区细胞，二是肝腺泡的1+2区细胞。所有细胞均通过激光捕获显微切割技术分离，并按照标准流程提取mRNA。