小鼠胚胎成纤维细胞复制性衰老模型的构建及衰老溶酶体功能检测  

Construction of a replicative senescence model of mouse embryonic fibroblasts and detection of lysosome function in senescent mouse embryonic fibroblasts

作　　者：朱海珍 姚铖铖 周涛[1] 李爱玲[1] 潘欣[1] ZHU Hai-zhen;YAO Cheng-cheng;ZHOU Tao;LI Ai-ling;PAN Xin(National Center of Biomedical Analysis,Beijing 100850,China)

机构地区：[1]国家生物医学分析中心,北京100850

出　　处：《军事医学》2023年第6期431-435,共5页Military Medical Sciences

基　　金：国家杰出青年科学基金项目(82025028)。

摘　　要：目的构建小鼠胚胎成纤维细胞(MEF)复制性衰老模型,检测衰老溶酶体相关功能改变,为衰老相关溶酶体疾病提供有效的细胞模型。方法通过酶消化法分离提取MEF,体外连续传代培养构建MEF复制性衰老模型,实时定量PCR(qPCR)检测衰老标志物p16和p21,衰老相关β⁃半乳糖苷酶(SA⁃β⁃gal)染色方法进一步验证衰老状态;通过溶酶体探针类染料LysoTracker Red DND⁃99探针及LysoSensor Yellow/Blue DND⁃160双激发探针追踪酸性溶酶体定性检测溶酶体酸碱性及定量检测溶酶体pH值;通过偶联自淬BODIPY®染料的牛血清白蛋白(DQ⁃BSA)检测衰老溶酶体的降解能力。结果qPCR和SA⁃β⁃gal染色结果显示,MEF复制性衰老模型构建成功。与年轻P3代MEF相比,衰老P9代MEF溶酶体的酸性明显丧失,差异有统计学意义(P<0.0001),衰老P9代MEF溶酶体的降解能力明显降低(P<0.05)。结论成功构建MEF复制性衰老细胞模型,并证明衰老MEF溶酶体的酸性丧失以及降解能力下降。Objective To construct a replicative senescence model of mouse embryonic fibroblasts(MEFs)detect the functional changes related to aging lysosomes,and provide an effective cell model for aging⁃related lysosomal diseases.Methods MEFs were isolated using the enzyme digestion method,and continuously cultured to construct a replicative senescence model.The senescence⁃related genes p16 and p21 were detected by real⁃time quantitative PCR(qPCR).Senescence associatedβ⁃galactosidase(SA⁃β⁃gal)staining was used to find out more about the aging of MEFs.Acidic lysosomes were qualitatively detected with the LysoTracker Red DND⁃99 probe,and the pH value of lysosomes was quantitatively detected with the LysoSensor Yellow/Blue DND⁃160 double excitation probe.The ability of aging lysosomes to degrade was detected with self⁃quenched BODIPY®dye conjugates of bovine serum albumin(DQ⁃BSA).Results qPCR and SA⁃β⁃Gal staining showed that a replicative senescence model of MEFs was constructed.Compared with the young MEFs,the acidity of lysosomes of senescent MEFs was significantly decreased(P<0.0001),so was the ability of the aging lysosomes to degrade(P<0.05).Conclusion A replicative senescence model of MEFs is constructed,the acidity of lysosomes of senescent MEFs is lost and the degradation ability is decreased.

关 键 词：细胞衰老 小鼠胚胎成纤维细胞 溶酶体 酸化 细胞模型 

分 类 号：R339.38[医药卫生—人体生理学] Q25[医药卫生—基础医学]