不同年龄人角化细胞的衰老特征评价  

Characteristics of Aging in Human Keratinocytes from Different Age Groups

作　　者：杨婷婷[1] 陈冬梅[1] 刘淑丹[1] 马海滨[1] 马晓娜[1] 梁雪云[1] Yang Tingting;Chen Dongmei;Liu Shudan(Human Stem Cell Institute of General Hospital,Ningxia Medical University,Ningxia 750004,China)

机构地区：[1]宁夏医科大学总医院干细胞研究所,银川750004

出　　处：《医学研究杂志》2018年第12期83-87,95,共6页Journal of Medical Research

基　　金：宁夏回族自治区自然科学基金资助项目(NZ15133)

摘　　要：目的评价年龄对衰老细胞模型的影响,以建立体外培养的衰老细胞模型。方法本研究根据供者年龄分为4组,每组3例生物学重复,<10岁、10～20岁、20～30岁和>30岁组。酶消化法体外分离培养不同年龄供体的表皮角化细胞。体外培养至P2代,进行以下检测:β-半乳糖苷酶染色鉴定衰老细胞数量;CCK-8和克隆形成试验检测细胞生长和增殖能力;磷酸化γ-H2A.X免疫荧光染色检测细胞DNA损伤;Western blot法检测分析细胞中周期蛋白依赖性激酶抑制蛋白表达。结果各组衰老细胞数占总细胞数百分比分别为0.864%、0.789%、5.472%、8.765%,差异有统计学意义(P=0.000)。随着年龄的增加,HKCs细胞倍增能力下降,倍增时间延长。<10岁组中细胞核Phospho-Histone H2A.X表达细胞数量少,>30岁组人正常角化细胞内DNA双链断裂大量产生。同时,细胞周期蛋白依赖性激酶抑制蛋白2A(CDKN2A/p16^(INK4a))、p21、p53的表达随年龄增加而上调,差异有统计学意义(P<0.05)。结论正常人角化细胞的增殖与衰老与人类年龄呈显著正相关,可以作为研究人类衰老的细胞模型。Objective Increased life spans have created a need for greater understanding of the diseases of old age including the integumentary system -the skin.In this study,The aging cell model was established by the analysis of the influence of age on the aging of normal keratinocytes.Methods Skin keratinocytes were isolated from different age donor by enzyme digestion method and cultured in vitro till P2 generation.Beta galactose glucoside enzyme staining detected the number of senescent cells using visible imaging.The growth and proliferation of keratinocytes was detected by CCK -8 assay and clone formation test.DNA damage was detected by immunofluorescence assay with a DNA damage marker γ-H2A.X.Cyclin-dependent kinase inhibitor protein of protein expression were analyzed by Western blot.Results Results showed that accumulation of senescent cells with age.The percentage of positive cells expressed SA -β - Gal was 0.864%,0.789% 5.472% and 8.765% within each group,respectively.The difference was statistically significant(P=0.000).Multiplication of the cell from aging donor gradually decreased,doubling time was gradually prolonged.Relatively small number of cell were induced to express the markers of DNA damage phospho -histone H2A in less than ten years old group.However,A large number of normal keratinocytes of DNA double strand fractures were produced in over 30 years of age group.The expression of cyclin -dependent kinase inhibitor protein p^16INK4a,p21 and p53 were significantly increased with age(P <0.05).Conclusion The proliferation and senescence of the normal keratinocytes are positively correlated with the age of human beings and can be used to study the aging of human beings as a cell model.

关 键 词：年龄相关衰老 角化细胞 细胞模型 增殖 DNA损伤 

分 类 号：R33[医药卫生—人体生理学]