肝星状细胞特异性Grk2基因敲除小鼠模型的制备及鉴定

Establishment and genotype identification of hepaticstellate cell-specific Grk2 gene knockout mouse model

目的利用Cre-loxP基因敲除技术建立肝星状细胞特异性G蛋白偶联受体激酶2(G protein-coupled receptor kinase 2,GRK2)基因敲除小鼠模型,为研究GRK2在肝星状细胞中的生物学功能提供动物模型基础。方法将loxP标记的Grk2基因小鼠(Grk2^(fl/fl))和Lrat-Cre工具鼠进行多次繁殖,建立肝星状细胞特异性Grk2基因敲除(Grk2^(ΔHSC))小鼠模型。观察和分析小鼠的生长繁殖情况;通过PCR反应鉴定flox和Cre基因型;免疫荧光双染检测肝星状细胞中GRK2表达;Western blot检测小鼠肝星状细胞及肺、脾、肾脏、心脏组织中GRK2蛋白表达;HE染色观察肝脏及肺、脾、心脏、肾脏组织学形态。结果成功鉴定Grk2^(ΔHSC)小鼠基因型;两组小鼠体质量、繁殖能力无明显差异;免疫荧光双染及Western blot结果表明,Grk2^(ΔHSC)小鼠的肝星状细胞中GRK2蛋白水平明显低于对照组小鼠,Grk2^(ΔHSC)小鼠肺、脾、肾脏和心脏组织中GRK2蛋白表达与对照组相比无明显变化;HE染色结果显示,Grk2^(ΔHSC)小鼠肝脏及主要组织结构与Grk2^(fl/fl)相比差异无显著性,可用于后续研究。结论本研究应用Cre-loxP技术成功构建了肝星状细胞特异性Grk2基因敲除小鼠,为进一步研究GRK2在肝脏中的作用提供了优良工具。

Aim To establish a stable hepatic stellate cell(HSC)-specific G protein-coupled receptor kinase 2(GRK2)knockout mice and provide the important animal model for further studying the biological function of GRK2 in HSC.Methods The loxP-labeled Grk2 gene mouse(Grk2^(fl/fl))and the Lrat-Cre mouse were hybridized to establish a HSC-specific Grk2 gene knockout mouse(Grk2^(ΔHSC))model.The growth and reproduction of mice were observed and analyzed.The flox and Cre gene were amplified by PCR for genotyping.Immunofluorescence double staining was used to examine the expression of GRK2 in HSC.Western blot was used to examine the expression of GRK2 in primary HSC,lung,spleen,kidney and heart tissue.HE staining was used to observe the structural changes in mice liver,lung,spleen,heart and kidney tissues.Results The genotypes of Grk2^(ΔHSC) mice were successfully identified.No significant differences were detected in diet,drinking water,reproductive ability between the two groups.The results of immunofluorescence double staining and Western blot showed that the expression of GRK2 in HSC was significantly lower in the Grk2^(ΔHSC) mice than that in the control mice,and the expression of GRK2 in other major tissue such as lung,spleen,kidney and heart of Grk2^(ΔHSC) mice had no significant changes compared with the control group.HE staining showed that there were no significant differences in structures of liver and other main tissue,which could be used for the follow-up study.Conclusions This study successfully establishes HSC specific Grk2 gene knockout mice using Cre-loxP technology,providing an excellent tool for further studying the role of GRK2 in liver.