猪成熟脂肪细胞分离培养及去分化研究

成熟脂肪去分化技术可为研究脂肪细胞分化提供均一的前体脂肪细胞。本研究分离培养了猪成熟脂肪细胞,并去分化为前体脂肪细胞。本实验采用Ⅱ胶原酶消化后离心分离1~3日龄仔猪(Susscrofa)皮下脂肪组织,天花板法培养获得成熟脂肪细胞。显微镜下观察脂肪细胞去分化形态学变化,并在成脂诱导培养液的作用下诱导再分化。采用油红O染色法检测分化不同时期细胞脂滴聚集效率,脂滴的累积随诱导的进行不断增加。RT-PCR检测成熟脂肪细胞标志基因过氧化物酶体增殖物活化受体(PPARγ)和和脂肪酸结合蛋白4(FABP4)的mRNA相对表达量,分化早期基因表达水平较低,其表达水平在分化过程中持续增高,在分化后期PPARγ相对表达量与诱导分化前增加了2.8倍,FABP4增加了约62倍(差异显著P<0.05)。说明去分化获得的前体脂肪细胞在成脂诱导培养液作用下,可有效地分化为成熟脂肪细胞。本研究优化了猪成熟脂肪细胞分离和培养体系,并通过去分化获得具有再分化能力的前体脂肪细胞,为进一步深入研究猪脂肪细胞分化与代谢提供技术平台。

Optimizing Cultivation of Mature Adipocytes and Obtaining Preadipocytes by Mature Adipocyte Dedifferentiation in Pigs(Sus scrofa)

The mature adipocyte dedifferentiation is able to provide homogeneous preadipocyees for researching adpocyte differentiation. In this study, mature adipocytes were isolated and cultured from adipose tissue, and then dedifferentiated to achieve preadipocytes. The subcutaneous adipose tissue from 3-day-old piglets(Sus scrofa) were digested by collagenase type Ⅱ, followed by centrifugation at different centrifugal force and then cultured by "ceiling culture" method. Morphological changes of adipocytes were observed under microscope, and the degree of adipogenesis and differentiation was assessed via oil red O staining. By inducing, adipose-derived progeny cells were redifferentiated into lipid-laden cells with accumulation of lipids. In terms of expression level, peroxisome proliferator-activated receptor-γ (PPARγ) and fatty acid binding protein 4 (FABP4) were detected by Real-time PCR. As we expected, the adipogenic markers, PPARγ and FABP4, increased along with the redifferentiation process. Particularly, a 2.8-fold increase of PPARγ expression, and a 62-fold of FABP4 were shown in the late phase of the redifferentiation process, demonstrating the significantly higher expression levels comparing with the unredifferentiated cells at 0 d(P<0.05). Showing that the predipocytes obtained by dedifferentiation might effectively differentiate to mature adipocytes with adipogenic induction agent. In addition, our study optimized the mature adipocytes culture system as well. Using this system, mature adipocytes can revert into proliferative-competent progeny cells by inducing redifferentiate again into mature adipocytes, which contributes an in vitro model for adipocytes investigation.