摘要
目的建立人网膜前脂肪细胞原代培养的方法,研究内脏脂肪增生肥大和内分泌功能的生物学特性。方法选取人网膜脂肪组织,采用酶消化细胞原代培养出梭形细胞,对培养细胞进行形态学观察、MTT 法测定生长曲线、油红 O 脂肪染色法进行脂肪细胞鉴定、酶联免疫法测定脂肪细胞因子瘦素和脂联素水平。结果培养出的梭形细胞成分均一。第3天开始增殖,4～9 d 为指数增长期,倍增时间约为60 h。经分化诱导21d 约有90%的细胞分化为成熟脂肪细胞。前脂肪细胞可检出低水平瘦素分泌,经诱导分化分泌量持续增多,第17天达峰值(1.6 ng·ml^(-1)·24 h^(-1)),之后保持高分泌状态至诱导结束;而脂联素直到诱导分化第7天始可检测到低水平分泌,此时光镜下已可见胞质含脂滴的细胞;7～17d 分泌逐渐增多,第17天分泌达峰值(99 ng·ml～·24 h^(-1)),之后有明显下降趋势。经油红 O 脂肪染色和瘦素、脂联素分泌检测证实分离培养的细胞为功能活跃的前脂肪细胞。结论成功建立了人网膜前脂肪细胞模型,观察到瘦素和脂联素不同分泌模式。脂联素可作为鉴定前脂肪细胞分化成熟的内分泌功能特异标志物。
Objective To develop a primary culture method of human omental preadipocytes and to study their biological properties, such as hyperplasia, hypertrophy and endocrine secretion of human visceral adipose tissue. Methods Using enzyme-digesting method, fibroblast-like cells from the human omental adipose tissues were cultured. The morphological changes of the cultured cells were observed and the growth curve was drawn by MTT method. The intracytoplasmic lipid of the cultured cells was determined by oil red O staining. The leptin and adiponectin levels in the culture supematants were measured by ELISA. Results The cultured fibroblast-like cells were homogeneous. Proliferation of cells began at the 3 rd day and the cell numbers increased in indicial way from the 3 rd day to the 9 th day. The doubling time of cells was about 60 hours. During the process of induction by conditional medium, the cells became round and larger, and more adipose droplets were aggregated. On the 21 st day, more than 90% of the cells became adipecytes. Leptin secretion was detected at low level in the preadipocytes and continuously increased during differentiation, with a peak on day 17. It remained constant from day 17 onward. Unlike leptin, adiponectin secretion was not detected until day 7 after induction, when differentiated adipecytes had already been observed. Its secretion increased dramatically between days 7 and 17, and reached a maximum level on day 17, but had a significant reduction on day 21. Extraction of intracytoplasmic lipid stained with oil red O and detection of leptin and adiponectin both verified the isolated cells were preadipecytes functioning actively. Conclusion A human omental preadipecytes model has been established and different secretion patterns of leptin and adiponectin secretion related to preadipecyte differentiation has been characterized. Adiponectin may be proposed as a specific marker for preadipecyte differentiation.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2007年第12期838-841,共4页
National Medical Journal of China
基金
卫生部专项基金(2001-162)
北京市科技新星计划资助项目(2004A27)
关键词
前脂肪细胞
原代培养
瘦素
脂联素
Preadipecyte
Primary cell culture
Leptin
Adiponectin
