小胶质细胞Stat3基因条件性敲除小鼠的构建及鉴定  

Construction and characterization of mice with conditional knockout of Stat3 gene in microglia

作　　者：朱晓晨 谢欣宜 赵旭日 徐丽娜 何智妍[1] 周薇[1] ZHU Xiaochen;XIE Xinyi;ZHAO Xuri;XU Lina;HE Zhiyan;ZHOU Wei(Laboratory of Oral Microbiota and Systemic Disease,Shanghai Ninth People's Hospital,Shanghai Jiao Tong University School of Medicine,College of Stomatology,Shanghai Jiao Tong University,National Center for Stomatology,National Clinical Research Center for Oral Diseases,Shanghai Key Laboratory of Stomatology,Shanghai Research Institute of Stomatology,Shanghai 200125,China)

机构地区：[1]上海交通大学医学院附属第九人民医院口腔微生态与系统性疾病实验室,上海交通大学口腔医学院,国家口腔医学中心,国家口腔疾病临床医学研究中心,上海市口腔医学重点实验室,上海市口腔医学研究所,上海200125

出　　处：《上海交通大学学报（医学版）》2023年第6期689-698,共10页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家自然科学基金(81971299,82071112);上海市“科技创新行动计划”自然科学基金项目(22ZR1437500)。

摘　　要：目的·基于Cre-Loxp系统构建小胶质细胞Stat3基因条件性敲除小鼠并验证其敲除效率。方法·将Cx3cr1^(creERT2)与Stat3^(fl/fl)基因型小鼠多代交配繁育,直到获得足够数量的Stat3^(fl/fl);Cx3cr1^(creERT2)基因型小鼠,即条件敲除小鼠(conditional knockout,CKO);同窝Stat3^(fl/fl)基因型小鼠,即对照小鼠(wild type,WT)。利用小鼠组织提取DNA后通过聚合酶链式反应(polymerase chain reaction,PCR),结合特异性引物分别扩增的结果来判断小鼠基因型。在CKO及WT小鼠6周龄时通过腹腔注射他莫昔芬诱导小胶质细胞中Stat3基因的敲除;2周后,随机选取CKO小鼠(n=4)及WT小鼠(n=4),利用磁珠分选法(magnetic activated cell sorting,MACS)分选出小胶质细胞后通过实时荧光定量PCR(real-time PCR,RT-PCR)技术从基因水平检测基因敲除效率;通过脑组织切片免疫荧光染色标记小胶质细胞和STAT3蛋白观察小胶质细胞中STAT3的表达情况;利用流式细胞术分别检测STAT3在小胶质细胞及脾巨噬细胞中的变化。通过尼氏染色检测神经元细胞的情况;通过脑组织切片荧光染色标记小胶质细胞,观察皮层和海马区域小胶质细胞的情况;通过流式细胞术检测小胶质细胞表型。结果·成功繁育出CKO小鼠和WT小鼠。MACS后脑细胞中小胶质细胞的占比从10%提升到85%。RT-PCR结果显示,相对于WT小鼠,CKO小鼠小胶质细胞中Stat3的mRNA相对表达量为0.3317±0.0414,mRNA水平下调(P=0.001)。脑组织免疫荧光染色结果显示,CKO小鼠小胶质细胞中STAT3蛋白的荧光强度较WT小鼠弱。脑组织流式细胞术显示:WT小鼠小胶质细胞中STAT3的阳性细胞占比为(85.30±5.69)%;CKO小鼠小胶质细胞中STAT3的阳性细胞占比为(39.70±3.88)%,STAT3表达率下降(P=0.001)。脾组织流式细胞术显示,CKO小鼠与WT小鼠脾巨噬细胞中STAT3的阳性细胞占比的差异无统计学意义(P>0.05)。尼氏染色显示,CKO小鼠与WT小鼠神经元细胞无显著差异。�Objective·To construct mice with conditional knockout of Stat3 gene in microglia based on the Cre-Loxp system and validate their knockout efficiency.Methods·Cx3cr1^(creERT2)and Stat3^(fl/fl)genotypic mice were bred for conditional knockout mice(CKO)and Wild Type mice(WT).The mouse genotypes were determined by extracting DNA from mouse tissues through Polymerase Chain Reaction(PCR)combined with the amplification results of cre and flox primers.Stat3 knockdown was induced by intraperitoneal injection of tamoxifen in the CKO and WT mice at 6 weeks of age.The CKO mice(n=4)and WT mice(n=4)were randomly selected for the detection.After two weeks of observation,microglia cells were sorted out by Magnetic Activated Cell Sorting(MACS).Real-time PCR(RT-PCR)was used to detect gene knockout efficiency at the gene level.The expression of STAT3 in microglia was observed by brain immunofluorescence staining.The expression rate of STAT3 in microglia was detected by flow cytometry.The expression rate of STAT3 in macrophages of the spleen was detected by flow cytometry.The condition of neuronal cells was examined by Nissl staining.The condition of the microglia in the cortex and hippocampus was observed by brain immunofluorescence staining.The phenotype of the microglia was detected by flow cytometry.Results·The CKO mice and WT mice were successfully bred.MACS boosted the proportion of microglia in brain cells from 10%to 85%.RT-PCR results showed that mRNA levels of Stat3 were down-regulated in microglia of CKO mice,compared with the WT mice(P=0.001).The relative mRNA expression of Stat3 in microglia of the CKO mice was 0.3317±0.0414.Immunofluorescence staining of brain tissues showed that the fluorescence intensity of STAT3 in microglia of the CKO mice was weaker than that of the WT mice.Flow cytometry of brain tissues showed that the STAT3-positive cells in microglia of the WT mice was(85.30±5.69)%and the CKO mice was(39.70±3.88)%.STAT3 expression was decreased in microglia of the CKO mice(P=0.001).Flow cytometry of spleen

关 键 词：小胶质细胞 信号转导和转录激活因子3(STAT3) 他莫昔芬 小鼠 CRE重组酶 

分 类 号：R781[医药卫生—口腔医学]