正常人成纤维细胞体外培养过程中β-半乳糖苷酶的变化(英文)  

Changes of β-galactosidase in normal fibroblasts:In vitro culture study

作　　者：蔡霞[1] 高学军[2] 冯令军[2] 张鹏[1] 唐胜建[1] 

机构地区：[1]潍坊医学院整形外科研究所,山东省潍坊市261042 [2]潍坊医学院附属医院普外科,山东省潍坊市261041

出　　处：《中国临床康复》2005年第30期224-226,i0006,共4页Chinese Journal of Clinical Rehabilitation

基　　金：山东省教育厅重点资助项目(NO.03K07)~~

摘　　要：背景:二倍体成纤维细胞常用于研究细胞水平的衰老,β-半乳糖苷酶是鉴别衰老细胞的标志酶,本实验拟观察正常人成纤维细胞在体外培养过程中β-半乳糖苷酶的变化情况。目的:建立一个稳定的二倍体成纤维细胞体外培养体系,了解正常人成纤维细胞老化过程中β-半乳糖苷酶的变化规律。设计:自身对照实验。单位:潍坊医学院整形外科研究所、潍坊医学院附属医院普外科。材料:实验于2000-09/2002-09在潍坊医学院整形外科研究所完成。实验样本来源于在潍坊医学院附属医院普外科行包皮环切术的6～8岁正常男性儿童(n=20)切除的健康包皮组织。方法:①成纤维细胞培养:取包皮组织,分离真皮和表皮,真皮部分用含100mL/L胎牛血清DMEM培养液终止胰蛋白酶作用,再用200U/mLⅠ型胶原酶37℃条件下消化30min,收集细胞悬液,接种于培养皿中,细胞培养达80%汇合时进行传代培养。②细胞鉴定:用相差显微镜观察细胞形态变化及生长增殖情况、透射电镜及抗vimentin免疫细胞化学染色。③细胞生物学行为的观察对比:取生长状态良好的细胞,按群体倍增水平分为两组,第一组代表年轻细胞,采用10代龄;第二组代表老化细胞,采用65代龄。进行消化传代,分别按2×104,4×104分别接种于24孔培养板内。每隔24h消化计数,每次每组计数3孔,计算均值。共记数8d,以培养时间为横轴,细胞数为纵轴,绘制生长曲线并计算细胞的群体倍增时间。④β-半乳糖苷酶组化检测及阳性细胞的半定量分析:成纤维细胞每5n代龄(共13代)作细胞爬片,漂洗,加入SA-β-gal染色液,37℃孵育48h。在显微镜下观察计数,蓝染细胞为阳性细胞,每次随机挑选视野计数500个细胞,得到阳性细胞百分率。主要观察指标:①正常人成纤维细胞生物学行为的改变。②正常人成纤维细胞老化过程中β-半乳糖苷酶的表达。结果:①正常人成纤维细�BACKGROUND ：Diploid fibroblasts are commonly used in study of senescence at the cellular level, and β-galactosidase is the marker enzyme of cell aging. This experiment is aimed to observe the changes of β-galactosidase in fibroblasts during in vitro culture. OBJECTIVE： To establish a stable in vitro culture system for diploid β-broblasts and observe the changes of β-galactosidase in normal fibroblasts during the aging process. DESIGN： Self-control experiment. SETTING： Institute of Plastic Surgery, Weifang Medical College; Department of General Surgery, Affiliated Hospital of Weifang Medical College. MATERIALS： This experiment was carried out at the Institute of Plastic Surgery, Weifang Medical College between September 2000 and September 2002. Samples were obtained from normal prepuce tissues （n=20） of boys aged 6-8 years old who received peritomy at the Department of General Surgery, Affiliated Hospital of Weifang Medical College.Informed consent was obtained. METHODS： ①Fibroblast culture： The prepuce tissues were obtained, and then dermis and epidermis were separated, the latter was digested with trypsin. For the dermis, DMEM containing 100 mL/L FBS was used to terminate the reaction before digested with 200 U/mL collagenase I at 37℃ for 30 minutes, cells were collected and inoculated in the culture dish; when cell congregation reached 80%, they were digested and reinoculated in the new culture dish for subculture. ②Cell identification： The morphological changes and growth of cells were observed under the inverted phase contrast microscope; the transmission electron microscope and anti-vimentin immune cytochemieal staining were also used.③ Comparison of cytobiological behavior： The well-growing cells were divided into two groups according to the level of clone multiplication. The first group represented young cells, using 10 generations of age; the second group represented aged cells, using 65 generations of age. The cells were digested and inoculated in 24-well culture dishes

关 键 词：成纤维细胞 细胞 培养的 细胞衰老 β半乳糖苷酶类 

分 类 号：R318[医药卫生—生物医学工程]