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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 establish a method of primary culture of human retinal preadipocytes to study the biological characteristics of visceral fat hyperplasia and endocrine function. Methods The human omental adipose tissue was harvested, and the primary culture of spindle cells by enzyme digestion was performed. The cultured cells were observed morphologically. The growth curve was determined by MTT assay. The fat cells were identified by oil red O staining. Adipocytokines leptin and adiponectin levels. Results The cultured spindle cells were homogenous. Proliferation on day 3, exponential growth phase on day 4 to 9, doubling time was about 60 h. About 90% of the cells differentiated into mature adipocytes after 21d of differentiation induction. Preadipocytes could detect low level of leptin secretion, the number of induced differentiation and secretion continued to increase, reaching the peak on the 17th day (1.6 ng · ml -1 · 24 h -1), and then maintained high secretion Until the end of induction; while adiponectin until the induction of differentiation began to detect low levels of secretion on the first 7 days, this time under the microscope can be seen in the cytoplasm lipid droplets of cells; 7 ~ 17d secretion gradually increased, the first 17 days peaked ( 99 ng · ml ~ · 24 h ~ (-1)), followed by a significant downward trend. Oil red O staining and leptin, adiponectin secretion test confirmed that the cells isolated and cultured as preadipocytes with active function. Conclusion The model of pre-human omental preadipocytes was successfully established and the different secretion patterns of leptin and adiponectin were observed. Adiponectin can be used as an endocrine-function-specific marker to identify preadipocyte maturation.