Kang Xihai, Xie Yubing, Kniss Douglas A
Department of Obstetrics and Gynecology, Laboratory of Perinatal Research, Ohio State University, College of Medicine and Public Health, Columbus, 43210, USA.
Tissue Eng. 2005 Mar-Apr;11(3-4):458-68. doi: 10.1089/ten.2005.11.458.
A better understanding of the mechanism of adipose tissue differentiation is of paramount importance in the development of therapeutic strategies for the treatment and prevention of obesity and type 2 diabetes mellitus. Optimal results using tissue culture models can be expected only when the in vitro adipocyte resembles adipose tissue in vivo as closely as possible. In this study, we used tissue-engineering principles to develop a three-dimensional (3-D) culture system to mimic the geometry of adipose tissue in vivo. Mouse preadipocyte 3T3-L1 cells were seeded onto nonbiodegradable fibrous polyethylene terephthalate scaffolds and differentiated with a hormone cocktail consisting of insulin, dexamethasone, isobutylmethylxanthine, and fetal calf serum. Cell morphology, growth, differentiation, and function were studied by immunocytochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, enzyme-linked immunosorbent assay, and oil red O staining. Cells grown on 3-D fibrous scaffolds were differentiated in situ by hormone induction with high efficiency (approximately 90%) as shown by scanning electron microscopy. Immunocytochemistry, immunoblot analysis, and RT-PCR revealed that the 3-D constructs expressed adipocyte-specific genes, including peroxisome proliferator-activated receptor gamma, leptin, adipsin, aP2, adiponectin, GLUT4, and resistin. Adipocytes matured on 3-D constructs secreted leptin at levels even greater than that of fully differentiated adipocytes in 2-D conventional cell cultures. Finally, adipocyte-specific phenotypic function was demonstrated by accumulation of neutral lipids in larger fat droplets. In conclusion, preadipocytes grown on 3-D matrices acquire morphology and biological features of mature adipocytes. This new culture model should have significant utility for in vitro studies of adipocyte cell biology and development.
更好地理解脂肪组织分化机制对于制定治疗和预防肥胖症及2型糖尿病的治疗策略至关重要。只有当体外脂肪细胞尽可能接近体内脂肪组织时，使用组织培养模型才能获得最佳结果。在本研究中，我们运用组织工程原理开发了一种三维（3-D）培养系统，以模拟体内脂肪组织的几何结构。将小鼠前脂肪细胞3T3-L1接种到不可生物降解的纤维状聚对苯二甲酸乙二酯支架上，并用由胰岛素、地塞米松、异丁基甲基黄嘌呤和胎牛血清组成的激素混合物进行分化。通过免疫细胞化学、逆转录聚合酶链反应（RT-PCR）、蛋白质印迹法、酶联免疫吸附测定和油红O染色研究细胞形态、生长、分化和功能。扫描电子显微镜显示，在3-D纤维支架上生长的细胞通过激素诱导原位高效分化（约90%）。免疫细胞化学、免疫印迹分析和RT-PCR显示，3-D构建体表达脂肪细胞特异性基因，包括过氧化物酶体增殖物激活受体γ、瘦素、脂肪酶、aP2、脂联素、葡萄糖转运蛋白4和抵抗素。在3-D构建体上成熟的脂肪细胞分泌瘦素的水平甚至高于二维传统细胞培养中完全分化的脂肪细胞。最后，通过中性脂质在较大脂滴中的积累证明了脂肪细胞特异性表型功能。总之，在3-D基质上生长的前脂肪细胞获得了成熟脂肪细胞 的形态和生物学特征。这种新的培养模型对于脂肪细胞生物学和发育的体外研究应具有重要的实用价值。
