鉴定血管加压素受体(V1aR)在小鼠肝脏枯否细胞和巨噬细胞中的表达

Identify the Expression of Vasopressin V1a Receptor (V1aR) in Mouse Liver Kupffer Cells and Macrophages

为了研究V1aR在肝脏枯否细胞和巨噬细胞RAW264.7中的表达,为今后研究V1aR表达与巨噬细胞功能间的关系奠定基础,本研究首先使用胶原酶灌注法和磁珠分选法(MACS)分离纯化小鼠肝脏枯否细胞;然后分别使用普通显微镜、透射电镜和流式细胞仪对分离纯化后枯否细胞的纯度与功能进行评估;最后使用反转录多聚酶链式反应(RT-PCR)、Western blot和免疫荧光检测V1aR在肝脏枯否细胞和巨噬细胞RAW264.7中的表达。普通显微镜和透射电镜结果表明本研究分离纯化后的桮否细胞具有巨噬样细胞的形态特征和吞噬活性;且流式细胞仪结果表明本研究分离纯化的枯否细胞的纯度可以达到98%以上。RT-PCR和Western blot结果表明在mRNA和蛋白质水平均能检测到V1aR在枯否细胞和巨噬细胞RAW264.7中的表达。同时免疫荧光实验进一步验证了V1aR在巨噬细胞RAW264.7中的表达。总之,本研究首次鉴定到了血管加压素受体V1aR在巨噬样细胞中的表达。

In ordrer to identify the expression ofvasopressin V1 a receptor （VlaR） in mouse liver Kupffer cells and mouse macrophages RAW264.7, which will facilitate the study of the relationship between the expression and function of VlaR in macrophages, follow methods were used in this study. Firstly, two-step collagenase perfusion and magnetic bead sorting （MACS） methods were used for isolation and purification of mouse Kuppfer cells. Secondly, general microscope, transmission electron microscopy, and flow cytometry were used to evaluate the purity and function of isolated Kupffer cells. Finally, reverse transcription-polymerase chain （RT-PCR）, Western blot, and immunofluorescence were used to detect the expression of V1 aR in Kupffer cells and macrophages RAW264.7. The results of general microscope and transmission electron microscopy showed that the isolated Kupffer cells have macrophage-like morphology and phagocytic function. The flow cytometer results showed that the purity of isolated Kupffer ceils in this study is higher than 98%. The expression of VlaR at mRNA and protein levels in Kupffer cells and RAW264.7 were detected using RT-PCR and Western blot, respectively. We also further confirmed the expression of VlaR in macrophages using immunofluorescence. In summary, the expression of VlaR was found in macrophage-like cells.