摘要
目的观察LMNAR527C基因突变纯合型小鼠体型、体质量、繁殖能力和生存期等表型,其组织、细胞衰老相关蛋白的表达情况,以探讨LMNAR527C基因突变对衰老的影响。方法LMNA基因杂合型小鼠6只,通过杂交方式得到LMNAR527C基因突变纯合型小鼠,比较野生型(基因型LMNAWT/WT)、杂合型(基因型LMNAWT/R527C)、纯合型(基因型LMNAR527C/R527C)小鼠体型、体质量、繁殖能力和生存期,采用Western blotting法检测不同基因型小鼠尾组织γH2Ax蛋白、免疫组化法检测皮肤组织γH2Ax蛋白。采用TRIzol提取正常和LMNA基因突变小鼠尾组织总RNA,PCR扩增小鼠LMNA和LMNAR527C突变基因,并与pEGFP-N1质粒重组获得pEGFP-N1-LMNA和pEGFP-N1-LMNA R527C重组质粒,然后转染293T LMNA敲除细胞(转染pEGFP-N1空质粒为空白组,转染pEGFP-N1-LMNA质粒为对照组,转染pEGFP-N1-LMNA R527C质粒为实验组),Western blotting法检测各组γH2Ax蛋白。将293T细胞设为正常组,免疫荧光法观察实验组LaminA蛋白的分布和定位。体外培养野生型和纯合型小鼠肺原代成纤维细胞,进行衰老相关β-半乳糖苷酶活性染色实验。结果三种不同基因型小鼠体型、体质量、繁殖能力和生存期比较,P均>0.05。设野生型小鼠γH2Ax蛋白相对表达量为1,纯合突变、杂合突变、野生型小鼠γH2Ax蛋白相对表达量分别为2.40±0.0432、1.72±0.0353,纯合突变、杂合突变型小鼠高于野生型小鼠(P均<0.05)。纯合突变型小鼠、野生型小鼠γH2Ax阳性细胞百分比分别为38%±2.25%、17%±1.68%,二者比较,P<0.05。与空白组比较,对照组和实验组γH2Ax蛋白水平升高(P均<0.05)。实验组细胞Lamin A蛋白容易在核膜处形成点状聚集,部分蛋白从核纤层转移至核内。纯合型、野生型小鼠肺原代成纤维细胞中β-半乳糖苷酶活性染色阳性细胞率分别为11.0%±1.96%、2.5%±0.78%,二者比较,P<0.05。结论LMNAR527C基因纯合突变小鼠未发现明显衰老表型。L
Objective To observe the phenotypes of body type,body mass,reproductive capacity and survival of mice with homozygous LMNAR527C gene mutation,and to observe the expression of senescence-related proteins in tissues and cells for the sake of exploring the effect of LMNAR527C gene mutation on aging.Methods Homozygous LMNAR527C gene mutation mice were obtained by hybridization of six LMNAR527C gene heterozygous mice,then we compared the body type,body mass,reproductive capacity and survival duration between the wild type(genotype LMNAWT/WT),heterozygous(genotype LMNAWT/R527C),homozygous(genotype LMNAR527C/R527C)mice,and theγH2Ax protein in the tail tissues of different genotype mice was detected by Western blotting,and theγH2Ax protein in the skin tissues was detected by immunohistochemistry.We extracted the total RNA from tail tissues of normal and LMNA mutant mice by TRIzol,LMNA and LMNAR527C mutant genes were amplified by PCR,afterwards,we recombined pEGFP-N1 plasmid with the two amplified genes to get pEGFP-N1-LMNA and pEGFP-N1-LMNA R527C recombinant plasmids,and then they were used to transfect 293T LMNA knockout cells(using empty plasmid to transfect as the blank group,pEGFP-N1-LMNA plasmid as the control group,pEGFP-N1-LMNA R527C plasmid as the experimental group).We detectedγH2Ax protein by Western blotting,and observed the distribution and localization of LaminA protein in the experimental group when setting 293T cells as the control group.Wild type and homozygous mouse lung primary fibroblasts were cultured in vitro,and senescence-associatedβ-galactosidase activity staining experiments were performed.Results No significant differences were found in the body weight,body mass,reproductive capacity or survival time between these three distinct genotype mice(all P>0.05).As we set the relative expression ofγH2Ax protein in wild type mice was 1,the relative expression levels ofγH2Ax protein in homozygous mutant and heterozygous mutant mice were 2.40±0.0432 and 1.72±0.0353,respectively(P<0.05).The percenta
作者
李东明
刘恒
朱兰玉
方玲
吴华裕
潘尚领
林有坤
舒伟
LI Dongming;LIU Heng;ZHU Lanyu;FANG Ling;WU Huayu;PAN Shangling;LIN Youkun;SHU Wei(Basic Medical College of Guangxi Medical University,Nanning 530021,China)
出处
《山东医药》
CAS
2019年第33期5-9,共5页
Shandong Medical Journal
基金
国家自然科学基金资助项目(31660311)
广西研究生教育创新计划项目(YCSW2019103)
