巨噬细胞条件性GP73基因敲除小鼠的构建及鉴定  

Construction and identification of macrophage⁃conditional GP73 gene knockout mice

作　　者：黄庆 甄兰 赵桂霖 赵汝舟 叶新萍[3] 吴飞翔[1,4,5] HUANG Qing;ZHEN Lan;ZHAO Guilin;ZHAO Ruzhou;YE Xinping;WU Feixiang(Department of Hepatobiliary Surgery,Guangxi Medical University Cancer Hospital,Nanning 530021,China;Institute of Biotechnology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China;The First Hospital of Hunan University of Chinese Medicine,Changsha 410007,China;Guangxi Liver Cancer Diagnosis and Treatment Engineering and Technology Research Center,Nanning 530021,China;Key Laboratory of Early Prevention and Treatment Regional High Frequency Tumor(Guangxi Medical University),Ministry of Education,Nanning 530021,China)

机构地区：[1]广西医科大学附属肿瘤医院肝胆外科,南宁530021 [2]军事医学研究院生物工程研究所,北京100850 [3]湖南中医药大学第一附属医院体检中心,长沙410007 [4]广西肝癌诊疗工程技术研究中心,南宁530021 [5]区域性高发肿瘤早期防治研究教育部重点实验室(广西医科大学),南宁530021

出　　处：《中国癌症防治杂志》2023年第6期630-636,共7页CHINESE JOURNAL OF ONCOLOGY PREVENTION AND TREATMENT

基　　金：广西重点研发计划项目(桂科AB16380242);区域性高发肿瘤早期防治研究教育部重点实验室(广西医科大学)自主课题(GKE⁃ZZ202216)。

摘　　要：目的构建并鉴定巨噬细胞条件性高尔基体蛋白73(golgi protein 73,GP73)基因敲除小鼠,为进一步研究GP73调控巨噬细胞功能影响肿瘤性疾病的发生发展提供动物模型。方法基于Cre/LoxP重组系统构建GP73^(flox/+)小鼠,通过GP73^(flox/+)小鼠雌雄自交得到GP73flox/flox小鼠。将GP73flox/flox小鼠与Lyz2⁃Cre+小鼠杂交,得到GP73flox/+Lyz2⁃Cre+小鼠,再将其与GP73flox/flox小鼠杂交,最终得到基因型为GP73flox/floxLyz2⁃Cre+的巨噬细胞条件性GP73基因敲除小鼠(MKO小鼠);以GP73flox/floxLyz2⁃Cre-小鼠作为对照组小鼠(GP73^(fl/fl)小鼠)。采用PCR和琼脂糖凝胶电泳鉴定小鼠flox及Cre基因型。采用实时荧光定量PCR(qPCR)及蛋白免疫印迹(Western blot)分别从mRNA水平和蛋白水平验证小鼠巨噬细胞GP73敲除效果及组织特异性。计算小鼠各组织质量与体重的比值以分析小鼠的生长发育情况,并检测小鼠血生化指标。结果通过基因鉴定、mRNA水平及蛋白水平证实巨噬细胞条件性GP73基因敲除小鼠构建成功。qPCR检测显示,与GP73^(fl/fl)小鼠相比,MKO小鼠骨髓来源巨噬细胞(bone marrow⁃derived macrophages,BMDMs)和腹腔原位巨噬细胞(peritoneal macrophages,PM)中GP73 mRNA水平均降低(P<0.0001);Western blot检测结果显示,GP73蛋白在MKO小鼠BMDMs和PM中的表达水平均较GP73^(fl/fl)小鼠明显降低,但在肝脏、肾脏及胸腺组织中GP73蛋白表达水平无显著差异。与GP73^(fl/fl)小鼠相比,MKO小鼠心、肝、脾、肺、肾、棕色脂肪及白色脂肪组织重量与体重之比无差异,各组织无形态学差异。血生化检测结果显示,MKO小鼠与GP73^(fl/fl)小鼠的各项血生化指标差异均无统计学意义(P>0.05)。结论成功构建巨噬细胞条件性GP73基因敲除小鼠模型,为深入研究GP73调控巨噬细胞功能在肿瘤性疾病中的作用和机制提供良好的动物模型。Objective To construct and identify macrophage⁃conditional golgi protein 73(GP73)gene knockout mice,and to provide an animal model for exploring the effect of GP73 on the occurrence and development of neoplastic diseases by regulating the function of macrophages.Methods The GP73^(flox/+)mice were constructed based on the Cre/LoxP recombination system.GP73flox/flox mice were obtained by self⁃cross of GP73flox/+mice.GP73flox/flox mice were crossed with Lyz2⁃Cre+mice to obtain GP73flox/+Lyz2⁃Cre+mice,and then crossed with GP73flox/flox mice to obtain the macrophage⁃conditional GP73 gene knockout mice,referred to as GP73flox/floxLyz2⁃Cre+mice(MKO mice).The GP73flox/floxLyz2⁃Cre-(GP73^(fl/fl))mice were used as the control mice.The genotypes of flox and Cre were identified by PCR and agarose gel electrophoresis.Real⁃time fluorescence quantitative PCR(qPCR)and Western blot were used to verify the knockout efficiency and tissue specificity of macrophages GP73 at mRNA and protein levels,respectively.The ratio of tissue weight to body weight was calculated to analyze the growth and development of mice,and the blood biochemical indicators of mice were detected.Results The successful construction of macrophage⁃conditional GP73 gene knockout mice was confirmed by genotype identification,mRNA,and protein level analysis.qPCR tests showed that,compared with GP73^(fl/fl)mice,the GP73 mRNA levels in bone marrow⁃derived macrophages(BMDMs)and peritoneal macrophages(PM)of MKO mice were decreased(P<0.0001).Western blot detection showed that compared with GP73^(fl/fl)mice,the expression levels of GP73 protein in BMDMs and PM in MKO mice were significantly decreased,though no significant differences were found in the expression levels of GP73 protein in liver,kidney and thymus tissues.Compared with GP73^(fl/fl)mice,the ratio of body weight to tissue weight of heart,liver,spleen,lung,kidney,brown adipose and white adipose in MKO mice was not significantly different,and there was no significant morphological difference

关 键 词：高尔基体蛋白73 巨噬细胞 CRE/LOXP重组系统 条件性敲除 基因型鉴定 小鼠模型 

分 类 号：R73-354[医药卫生—肿瘤]