【摘要】 目的 探讨利用Cre-LoxP基因敲除技术构建肝特异性NLRP3基因敲除小鼠模型的可能性及基因鉴定方法。方法 第一阶段将肝细胞特异性表达白蛋白启动子-Cre(Alb^Cre)重组酶的小鼠与NLRP3^flox/flox小鼠进行交配，经2代选育配种，获取基因型为NLRP3^flox/flox/Alb^Cre+/-的肝细胞特异性NLRP3基因敲除小鼠（肝细胞NLRP3敲除组）和基因型为NLRP3^flox/flox/Alb^Cre-/-的小鼠为同窝对照小鼠（同窝对照组）。第二阶段为大量繁殖阶段，将NLRP3^flox/flox/Alb^Cre+/-目的小鼠与NLRP3^flox/flox小鼠交配繁殖可快速获得大量实验用目的小鼠与同窝对照小鼠。小鼠编号后剪取鼠尾提取DNA，采用PCR法鉴定繁殖小鼠的基因表型；qPCR和Western blot检测小鼠肝组织中NLRP3 mRNA和蛋白表达水平，HE染色观察肝组织形态学改变，检测血清肝转氨酶、炎症因子。观察2组小鼠体质量变化、肝体比值及繁殖发育中的特殊情况。结果 F2代中目的小鼠基因表型符合理论结果 NLRP3^flox/flox/Alb^Cre+/-；肝细胞NLRP3敲除组小鼠肝组织中NLRP3 mRNA及蛋白表达水平显著低于同窝对照组（P<0.05）；肝细胞NLRP3敲除组小鼠NLRP3基因敲除后不影响生长发育及繁殖，小鼠体质量、肝体比值、肝组织形态、血清肝转氨酶、炎症因子与同窝对照组比较，差异均无统计学意义（P>0.05）。结论 采用Cre-LoxP基因敲除技术可成功构建肝细胞特异性NLRP3基因敲除小鼠模型，为下一步动物层面研究肝中NLRP3基因功能提供了重要的技术支持。更多还原

【Abstract】 Objective To explore the possibility and genetic identification method of constructing a hepatocyte-specific NLRP3 gene knockout mouse model by using Cre-LoxP system gene knockout technology.Methods Phase one: mice specifically expressing the albumin promoter-Cre(Alb^Cre) recombinase in hepatocytes were mated with NLRP3^flox/flox mice, and the hepatocyte-specific NLRP3 gene knockout mice with the genotype of NLRP3^flox/flox/Alb^Cre+/- （hepatocyte NLRP3 knockout group） and the control mice in the same litter with the genotype of NLRP3^flox/flox/Alb^Cre-/- （control group in the same litter） were obtained after two generations of selection and mating. The second stage was the mass reproduction stage. Mating NLRP3^flox/flox/Alb^Cre+/- target mice with NLRP3^flox/flox mice could quickly obtain a large number of experimental target mice and control mice in the same litter. The DNA was extracted from the tails of mice after numbering, and the offspring genotype was identified by PCR. qPCR and Western blot were used to detect the mRNA and protein expression levels of NLRP3 gene in the liver tissue. HE staining was used to observe the morphological changes in liver tissues,and serum liver transaminases and inflammatory factors were detected. The changes in body weight, liver-to-body ratio and special circumstances during reproduction and development of mice in the two groups were observed.Results The offspring genotype of the target mice in the F2 generation was consistent with theoretical result of NLRP3^flox/flox/Alb^Cre+/-. The mRNA and protein levels of NLRP3 in liver tissues of mice in the hepatocyte NLRP3 knockout group were significantly lower than those in the control group in the same litter（P<0. 05）. The mice in the hepatocyte NLRP3 knockout group was not affected in terms of growth, development and reproduction after the NLRP3 gene knockout. There were no statistically significant differences in the body weight, liver-to-body ratio, liver tissue morphology, serum liver transaminase or inflammatory factors between the hepatocyte NLRP3 knockout group and the control group in the same litter（P>0. 05）. Conclusion The Cre-LoxP gene knockout technology can be used to successfully construct a hepatocyte-specific NLRP3 gene knockout mouse model, providing an important technical support for the next step of studying the function of the NLRP3 gene in the liver at the animal level.更多还原