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

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正常人成纤维细胞生物学行为的改变:细胞生长曲线显示,老化细胞生长速度较年轻细胞明显减慢。年轻细胞最大增殖数约为47.3×105,对数生长期在3.0～6.0d,细胞倍增时间为2.18d。老化细胞最大增殖数各为8.5×104,对数生长期分别在4.0～7.5d,细胞群体倍增时间依次为3.86d。②正常人成纤维细胞老化过程中β-半乳糖苷酶的表达:与年轻细胞组(阳性细胞率=4%)相比,衰老细胞组中阳性细胞率(60%)大为增强。直线相关分析结果显示,β-半乳糖苷酶染色的阳性率和细胞代龄之间呈显著正相关(r=0.92,P<0.01)。结论:在正常人成纤维细胞从年轻向老化发展的过程中,细胞群体倍增时间呈增高的趋势;与年轻细胞相比较(占4%),β-半乳糖苷酶的表达在老化细胞中显著增强(占60%),且这种增强与细胞衰老表型的出现和细胞增殖能力的丧失相平行,可反应细胞的老化程度。本实验为建立成纤维细胞衰老模型提供了实验依据,同时也为老年学及肿瘤生物学甚至组织工程学发展提供科学依据。

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 by 2×10^4 and 4×10^4, respectively. Cells were digested and counted once every 24 hours, 3 wells in each group were counted each time for calculating the average, altogether for 8 days. Taking the culture days as the abscissa axis and cell number as the ordinate axis, growth curve was drawn and population double time was calculated.④Histochemieal examination of β-galactosidase and semi-quantitative analysis of positive cells： Fibroblasts were used for slide attaching by every 5n generation age , rinsed and stained with SA-β-gal at 37℃ for 48 hours. Blue colored positive cells were counted under the microscope to calculate the percentage of positive cells in randomly selected 500 cells within one field of vision, which represented cell aging rate, MAIN OUTCOME MEASURES： ①Biologieal behavioral changes of normal human fibroblasts;②Expression of β-galaetosidase in normal human fibroblasts during the aging process. RESULTS： ①Biological behavioral changes of normal fibroblasts： According to the cell growth curve, aging cells grew obviously more slowly than the younger ones. The maximum multiplication value of young cells was 47.3×10^5, with logarithm growth period varying within 3.0-6.0 days and cell multiplication time of 2.18 days, as compared to the corresponding 8.5×10^4, 4.0-7.5 days and 3.86 days of aging cells.② Expression of β-galactosidase in normal human fibroblasts during the aging process： The positive cell rate in aging cell group greatly increased （60%） as compared to that of young cells （4%）. The li.near correlation analysis revealed that β-galactosidase positive cells had remarkably positive correlation with cell generation age （r=0.92, P 〈 0.01）. CONCLUSION： Cell clone multiplication time was found on an increasing tendency with normal fibroblasts developing from young to senile. In contrast with that of young cells （4%）, the expression of β-galaetosidase in aging cells （60%） was remarkably enhanced, which paralleled with the appearance of cell senile phenotype and cell proliferation, and can be used to reflect the degree of cell aging. This experiment provides experimental basis for the establishment of fibroblast aging model; meanwhile it also provides scientific evidence for the development of tumor biology and organic engineering.