肝细胞特异性Sirt3基因敲除小鼠模型的构建  

Construction of hepatocyte-specific Sirt3 gene knockout mouse model

作　　者：许雅萍 王语涵 陈婷婷 李南 高萍萍 李玲 王华[2] 孙妩弋[1] Xu Yaping;Wang Yuhan;Chen Tingting;Li Nan;Gao Pingping;Li Ling;Wang Hua;Sun Wuyi(Institute of Clinical Pharmacology,Anhui Medical University,Key Laboratory of Anti-inflammatory and Immune Drugs,Ministry of Education,Hefei 230032;Dept of Oncology,First Affiliated Hospital of Anhui Medical University,Hefei 230032)

机构地区：[1]安徽医科大学临床药理研究所,抗炎免疫药物教育部重点实验室,合肥230032 [2]安徽医科大学第一附属医院肿瘤科,合肥230032

出　　处：《安徽医科大学学报》2024年第3期384-390,共7页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金资助项目(编号:82370632);安徽省高校杰出青年科研项目(编号:2023AH020033);安徽省转化医学研究院科研基金项目(编号:2022zhyx-C07);安徽医科大学科研水平提升计划(编号:2021xkjT016);安徽医科大学第三附属医院基础与临床合作研究提升计划培育专项(编号:2022sfy014)。

摘　　要：目的运用Cre-loxP技术构建肝细胞特异性沉默信息调节因子3(silence information regulator 3,Sirt3)基因敲除(Sirt3^(Δhep))小鼠,为研究肝细胞Sirt3基因在疾病中的生物学功能提供重要动物模型。方法将loxP标记的Sirt3 ^(flox/flox)小鼠与Alb-Cre纯合子(Alb-Cre^(+/+))小鼠进行交配,F1代Sirt3^(flox/-)/Alb-Cre^(+/-)小鼠再与Sirt3^(flox/flox)小鼠进行交配并鉴定,F2代基因型为Sirt3 ^(flox/flox)/Alb-Cre^(+/-)的小鼠即为本实验所构建的Sirt3^(Δhep)小鼠,Sirt3 ^(flox/flox)/Alb-Cre^(-/-)小鼠即为对照小鼠Sirt3 ^(flox/flox)小鼠。提取鼠尾DNA,通过PCR鉴定子代小鼠的基因型;免疫荧光双染观察Sirt3在小鼠肝细胞中的表达;提取Sirt3^(Δhep)小鼠原代肝细胞及心脏、脾脏、肾脏、肺组织蛋白,Western blot法验证Sirt3在小鼠肝细胞及其他组织中的表达水平;HE染色观察小鼠肝脏及心脏、脾脏、肺等组织结构。结果成功鉴定出Sirt3^(Δhep)小鼠;免疫荧光及Western blot结果显示,小鼠肝细胞中Sirt3蛋白表达水平低于对照组小鼠(P<0.01),而Sirt3^(Δhep)小鼠的心脏、脾脏、肾脏和肺组织中Sirt3表达与对照组相比无明显变化(P>0.05);HE染色结果显示Sirt3^(Δhep)小鼠肝脏、心脏、脾脏、肺、肾脏的组织学特征与对照组小鼠相比无明显变化。结论成功构建肝细胞特异性Sirt3基因敲除小鼠,为进一步研究肝细胞Sirt3基因在相关疾病中的调控作用机制奠定了基础。Objective To construct hepatocyte-specific silence information regulator 3(Sirt3)gene knockout(Sirt3^(Δhep))mice by Cre-loxP technique,and to provide an important animal model for further studying the biological function of the hepatocyte Sirt3 gene in diseases.Methods LoxP-labeled Sirt3^(flox/flox) mice were mated with Alb-Cre homozygous(Alb-Cre^(+/+))mice,and the F1 generation Sirt3^(flox/-)/Alb-Cre^(+/-)mice were then mated with Sirt3^(flox/flox) mice,and the F2 genotype of Sirt3^(flox/flox)/Alb-Cre^(+/-)mice were the Sirt3^(Δhep) mice constructed in this experiment.Sirt3^(flox/flox)/Alb-Cre^(-/-)(Sirt3 ^(flox/flox))mice were the control mice.Mouse tail genome DNA was extracted and PCR was used to identify the genotypes of the offspring mice.Immunofluorescence was used to detect Sirt3 expression in mouse hepatocytes.Primary hepatocytes and tissue proteins of Sirt3^(Δhep) mice were extracted,and the expression of Sirt3 in mouse hepatocytes and other tissues was verified by Western blot.HE staining was used to observe mice′s liver,heart,spleen,and lung tissue structure.Results Sirt3^(Δhep) mice were successfully identified.Immunofluorescence and Western blot results demonstrated a significant decrease in the expression of Sirt3 in the hepatocytes of these mice compared to the control group(P<0.01).At the same time,there was no significant difference in the expression of Sirt3 in the heart,spleen,kidney,and lung tissues of Sirt3^(Δhep) mice compared with the control group(P>0.05).The results of HE staining showed that the histological characteristics of the liver,heart,spleen,lungs,kidneys,and other major organs of Sirt3^(Δhep) mice were not significantly different from those of the control group mice.Conclusion Hepatocyte-specific Sirt3 gene knockout mice are successfully constructed,which provides an animal model to explore further the role and molecular mechanism of the hepatocyte Sirt3 gene in diseases.

关 键 词：肝细胞 Sirt3 基因敲除 Sirt3^(Δhep)小鼠 CRE-LOXP 基因型鉴定 

分 类 号：R-332[医药卫生]